Odorant receptors

ABSTRACT

The invention provides methods and compositions relating to odorant receptors, including a general expression cloning methodology which is useful for identifying novel G protein-coupled receptors and a novel family of odorant receptors and related nucleic acids, ligands, agonists and antagonists. These compositions provide a wide variety of applications such as screening for related receptors, and by modulating the function of the disclosed receptors by modulating their expression or contacting them with agonists, antagonist or ligands modulating reproductive/sexual and non-sexual social behaviors mediated via the olfactory system, reproductive physiologies and olfactory system regulated feeding behaviors, migratory behaviors and events such as conception, implantation, estrous and menstruation.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority under 35UCS120 to U.S. Ser. No.09/619,353, filed Jul. 19, 2000, which claims priority under 35UCS120 toU.S. S No. 60/144,766, also entitled Odorant Receptors, filed Jul. 20,1999.

INTRODUCTION

[0002] 1. Field of the Invention

[0003] The field of the invention is odorant receptors.

[0004] 2. Background of the Invention

[0005] The detection and discrimination of the multitude ofenvironmental stimuli by the vertebrate olfactory system results fromthe activation of olfactory neurons within the olfactory epithelium ofthe nose (reviewed by Shepherd, 1994; Buck, 1996). The first step inolfactory processing resides at the level of the interaction of odorousligands with odorant receptors. A large multigene family thought toencode odorant receptors was initially identified in the rat (Buck andAxel, 1991). These receptors are predicted to exhibit a seventransmembrane domain topology characteristic of the superfamily of Gprotein-coupled receptors. The sizes of the receptor repertoires ofdifferent vertebrate species are extremely large and are estimated tocontain between 100 and 1000 individual genes (Buck, 1996). Theseobservations suggest that the initial step in olfactory discriminationis accomplished by the integration of signals from a large number ofspecific receptors, each capable of binding only a small number ofstructurally-related odorants. Consistent with this model, it has beenshown that one rat odorant receptor can be activated by 7 to 10 carbonn-aliphatic aldehydes (Zhao et al., 1997; see also Krautwurst et al.,1998; Malnic et al., 1999). In invertebrates, the C. elegans odr-10 geneencodes a G protein-coupled receptor that is sharply tuned to respond tothe odorant, diacetyl (Sengupta et al., 1996; Zhang et al., 1997).

[0006] Other olfactory G protein-coupled receptors unrelated to thereceptor gene family first described by Buck and Axel (1991) have beenidentified in the vomeronasal organ (VNO) of mammals (Dulac and Axel,1995; Herrada and Dulac, 1997; Matsunami and Buck, 1997; Ryba andTirindelli, 1997). The VNO is a specialization of the peripheralolfactory system in higher vertebrates that receives non-volatilepheromonal and non-pheromonal cues (Halpern, 1987). The VNO receptorsare encoded by two unrelated gene families; members of the VNR familyare localized in a subpopulation of VNO neurons defined by theirexpression of the G protein alpha subunit, Gai2 (Dulac and Axel, 1995;Berghard and Buck, 1996; Jia and Halpern, 1996), whereas members of theV2R family are expressed predominantly in a separate subpopulation ofGao-expressing cells (Herrada and Dulac, 1997; Matsunami and Buck, 1997;Ryba and Tirindelli, 1997). Interestingly, the V2R receptors arestructurally related to the calcium-sensing receptor (CaSR; Hebert andBrown, 1995) and metabotropic glutamate receptor (mGluR; Tanabe et al.,1992) families. While it has been proposed that both classes of VNOreceptors comprise pheromone receptors, the actual function of theseorphan receptors awaits a direct demonstration of their ligand bindingor ligand activation properties.

[0007] As an approach toward identifying ligands for olfactoryreceptors, we have pursued an expression cloning strategy using thegoldfish as a model system. Fish are thought to respond to a smallerrange of odorants than terrestrial vertebrates and thus appear topossess a smaller repertoire of odorant receptors (Ngai et al., 1993b).Moreover, the odorants that fish detect are water soluble, and includeamino acids (feeding cues), bile acids (nonreproductive social cues withpossible roles in migration), and sex steroids and prostaglandins (sexpheromonal cues) (reviewed by Hara, 1994; Sorensen and Caprio, 1998).Electrophysiological studies have defined the sensitivities of fisholfactory systems to specific ligands, demonstrating, for example,thresholds for detection in the picomolar (for sex steroids) tonanomolar (for amino acids) range (Hara, 1994). Thus, the definedproperties of odorant-evoked pathways in vivo provide an excellentstarting point for the molecular and biochemical characterization offish odorant receptors.

[0008] In the examples below, we describe the expression cloning of acDNA encoding a goldfish odorant receptor preferentially tuned torecognize basic amino acids. This cDNA encodes a G protein-coupledreceptor that shares significant similarity to receptor families thatinclude the CaSR, mGluR, and V2R class of VNO receptors. Degeneratepolymerase chain reaction (PCR) reveals other related sequences that areexpressed in the goldfish olfactory epithelium. Together our resultsindicate that these receptors comprise a family of odorant receptors.Moreover, the characterization of the goldfish amino acid receptor'sodorant tuning properties provides critical molecular parameters forconsidering models of molecular recognition and information coding inthe olfactory system.

[0009] Aspects of this invention have been published by Speca et al.(Neuron July 1999;23(3):487-98).

SUMMARY OF THE INVENTION

[0010] The invention provides methods and compositions relating toodorant receptors, including a general expression cloning methodologywhich is useful for identifying novel G protein-coupled receptors and anovel family of odorant receptors and related nucleic acids, ligands,agonists and antagonists. These compositions provide a wide variety ofapplications such as screening for related receptors, and by modulatingthe function of the disclosed receptors by modulating their expressionor contacting them with agonists, antagonist or ligands modulatingreproductive/sexual and non-sexual social behaviors mediated via theolfactory system, reproductive physiologies and olfactory systemregulated feeding behaviors, migratory behaviors and events such asconception, implantation, estrous and menstruation.

DESCRIPTION OF PARTICULAR EMBODIMENTS OF THE INVENTION

[0011] The following description of particular embodiments and examplesare offered by way of illustration and not by way of limitation. Whileparticularly directed and exemplified often in terms of goldfish R5.24,the following descriptions, including fragment limitations and assayutilizations, also apply to the other disclosed CaSR-like polypeptidesand polynucleotides.

[0012] The subject domains provide R5.24 domain specific activity orfunction, such as R5.24-mediated olfaction, ligand signal transducing ortransducing inhibitory activity and/or R5.24-specific bindingtarget-binding or binding inhibitory activity. R5.24-specific activityor function may be determined by convenient in vitro, cell-based, or invivo assays: e.g. in vitro binding assays, cell culture assays, inanimals (e.g. gene therapy, transgenics, etc.), etc. The specificbinding target may be a ligand, agonist or antagonist, a R5.24regulating protein or other regulator that directly modulates R5.24activity or its localization; or non-natural binding target such as aspecific immune protein such as an antibody, or a R5.24 specific agentsuch as those identified in screening assays such as described below.R5.24-binding specificity may be assayed by binding equilibriumconstants (usually at least about 10⁷ M⁻¹, preferably at least about 10⁸M⁻¹, more preferably at least about 10⁹ M⁻¹), by the ability of thesubject polypeptides to function as negative mutants in R5.24-expressingcells, to elicit R5.24 specific antibody in a heterologous host (e.g arodent or rabbit), etc.

[0013] Exemplary suitable R5.24 polypeptides (a) SEQ ID NO:02, or afunctional deletion mutant thereof or a sequence about 60-70%,preferably about 70-80%, more preferably about 80-90%, more preferablyabout 90-95%, most preferably about 95-99% similar to the R5.24 sequencedisclosed herein as determined by Best Fit analysis using defaultsettings and/or (b) is encoded by a nucleic acid comprising a naturalR5.24 encoding sequence (such as SEQ ID NO:01) or a fragment thereof atleast 36, preferably at least 72, more preferably at least 144, mostpreferably at least 288 nucleotides in length which specificallyhybridizes thereto. Suitable deletion mutants are readily screened inR5.24 binding or activation assays as described herein. Preferred R5.24domains/deletion mutants/fragments comprise at least 8, preferably atleast 16, more preferably at least 32, most preferably at least 64consecutive residues of SEQ ID NO:2 and provide a R5.24 specificactivity, such as R5.24-specific antigenicity and/or immunogenicity,especially when coupled to carrier proteins. The subject domains provideR5.24-specific antigens and/or immunogens, especially when coupled tocarrier proteins. For example, peptides corresponding to R5.24-specificdomains are covalently coupled to keyhole limpet antigen (I<LH) and theconjugate is emulsified in Freunds complete adjuvant. Laboratory rabbitsare immunized according to conventional protocol and bled. The presenceof R5.24-specific antibodies is assayed by solid phase immunosorbantassays using immobilized R5.24 polypeptides. R5.24 specific antigenicand/or immunogenic peptides encompass diverged sequence regions,preferably diverged extracellular or cytosolic regions, as seen inalignments with related sequences human CaSR, Fugu Ca02.1, mouse V2R2and rat mGluR1.

[0014] Suitable natural R5.24 encoding sequence fragments are of lengthsufficient to encode such R5.24 domains. In a particular embodiment, theR5.24 fragments comprise species specific fragments; such fragments arereadily discerned from alignments. Exemplary such R5.24-1 immunogenicand/or antigenic peptides are shown in Table 1. TABLE 1 ImmunogenicR5.24-1 polypeptides eliciting RS.24-1 specific rabbit polyclonalantibody: R5.24 polypeptide-KLH conjugates immunized per protocoldescribed above. R5.24 Polypeptide Immunogenicity SEQ ID NO:02, res.1-10 +++ SEQ ID NO:02, res. 29-41 +++ SEQ ID NO:02, res. 75-87 +++ SEQID NO:02, res. 92-109 +++ SEQ ID NO:02, res. 132-141 +++ SEQ ID NO:02,res. 192-205 +++ SEQ ID NO:02, res. 258-269 +++ SEQ ID NO:02, res.295-311 +++ SEQ ID NO:02, res. 316-330 +++ SEQ ID NO:02, res. 373-382+++ SEQ ID NO:02, res. 403-422 +++ SEQ ID NO:02, res. 436-442 +++ SEQ IDNO:02, res. 474-485 +++ SEQ ID NO:02, res. 502-516 +++ SEQ ID NO:02,res. 561-576 +++ SEQ ID NO:02, res. 595-616 +++ SEQ ID NO:02, res.640-656 +++ SEQ ID NO:02, res. 683-697 +++ SEQ ID NO:02, res. 717-732+++ SEQ ID NO:02, res. 768-777 +++ SEQ ID NO:02, res. 798-813 +++ SEQ IDNO:02, res. 829-843 +++ SEQ ID NO:02, res. 844-877 +++ SEQ ID NO:02,res. 852-875 +++

[0015] In one embodiment, the R5.24 polypeptides are encoded by anucleic acid comprising SEQ ID NO:01 or a fragment thereof whichhybridizes with a full-length strand thereof, preferably under stringentconditions. Such nucleic acids comprise at least 36, preferably at least72, more preferably at least 144 and most preferably at least 288nucleotides of SEQ ID NO:01. Demonstrating specific hybridizationgenerally requires stringent conditions, for example, hybridizing in abuffer comprising 30% formamide in 5×SSPE (0.18 M NaCl, 0.01 M NaPO₄, pH7.7, 0.001 M EDTA) buffer at a temperature of 42° C. and remaining boundwhen subject to washing at 42° C. with 0.2×SSPE (Conditions I);preferably hybridizing in a buffer comprising 50% formamide in 5×SSPEbuffer at a temperature of 42° C. and remaining bound when subject towashing at 42° C. with 0.2×SSPE buffer at 42° C. (Conditions II).Exemplary nucleic acids which hybridize with a strand of SEQ ID NO:01are shown in Table 2. TABLE 2 Exemplary nucleic acids which hybridizewith a strand of SEQ ID NO:01 under Conditions I and/or II. R5.24Nucleic Acid Hybridization SEQ ID NO:01, nucl. 1-47 + SEQ ID NO:01,nucl. 58-99 + SEQ ID NO:01, nucl. 95-138 + SEQ ID NO:01, nucl. 181-220 +SEQ ID NO:01, nucl. 261-299 + SEQ ID NO:01, nucl. 274-315 + SEQ IDNO:01, nucl. 351-389 + SEQ ID NO:01, nucl. 450-593 + SEQ ID NO:01, nucl.524-546 + SEQ ID NO:01, nucl. 561-608 + SEQ ID NO:01, nucl. 689-727 +SEQ ID NO:01, nucl. 708-737 + SEQ ID NO:01, nucl. 738-801 + SEQ IDNO:01, nucl. 805-854 + SEQ ID NO:01, nucl. 855-907 + SEQ ID NO:01, nucl.910-953 + SEQ ID NO:01, nucl. 1007-1059 + SEQ ID NO:01, nucl.1147-1163 + SEQ ID NO:01, nucl. 1258-1279 + SEQ ID NO:01, nucl.1375-1389 + SEQ ID NO:01, nucl. 1581-1595 + SEQ ID NO:01, nucl.1621-1639 + SEQ ID NO:01, nucl. 1744-1755 + SEQ ID NO:01, nucl.1951-1969 +

[0016] A wide variety of cell types express R5.24 polypeptides subjectto regulation by the disclosed methods, including many neuronal cells,transformed cells, infected (e.g. virus) cells, etc. Ascertaining R5.24binding or activation is readily effected by binding assays or cellsfunction assays as disclosed herein. Accordingly, indications for thesubject methods encompass a wide variety of cell types and function,etc. The target cell may reside in culture or in situ, i.e. within thenatural host.

[0017] In another aspect, the invention provides methods of screeningfor agents which modulate R5.24-ligand interactions. These methodsgenerally involve forming a mixture of a R5.24-expressing cell, a R5.24ligand and a candidate agent, and determining the effect of the agent onthe R5.24-ligand interaction. The methods are amenable to automated,cost-effective high throughput screening of chemical libraries for leadcompounds. Identified reagents find use in the pharmaceutical industriesfor animal and human trials; for example, the reagents may bederivatized and rescreened in in vitro and in vivo assays to optimizeactivity and minimize toxicity for pharmaceutical development.

[0018] The amino acid sequences of the disclosed R5.24 polypeptides areused to back-translate R5.24 polypeptide-encoding nucleic acidsoptimized for selected expression systems (Holler et al. (1993) Gene136, 323-328; Martin et al. (1995) Gene 154, 150-166) or used togenerate degenerate oligonucleotide primers and probes for use in theisolation of natural R5.24-encoding nucleic acid sequences (“GCG”software, Genetics Computer Group, Inc, Madison Wis.). R5.24-encodingnucleic acids are used in R5.24-expression vectors and incorporated intorecombinant host cells, e.g. for expression and screening, etc.

[0019] The invention also provides nucleic acid hybridization probes andreplication/amplification primers having a R5.24 cDNA specific sequencecomprising a fragment of SEQ ID NO: 1, and sufficient to effect specifichybridization thereto. Such primers or probes are at least 12,preferably at least 24, more preferably at least 36 and most preferablyat least 96 nucleotides in length. Demonstrating specific hybridizationgenerally requires stringent conditions, for example, hybridizing in abuffer comprising 30% formamide in 5×SSPE (0.18 M NaCl, 0.01 M NaPO₄, pH7.7, 0.001 M EDTA) buffer at a temperature of 42° C. and remaining boundwhen subject to washing at 42° C. with 0.2×SSPE; preferably hybridizingin a buffer comprising 50% formamide in 5×SSPE buffer at a temperatureof 42° C. and remaining bound when subject to washing at 42° C. with0.2×SSPE buffer at 42° C. R5.24 nucleic acids can also be distinguishedusing alignment algorithms, such as BLASTX (Altschul et al. (1990) BasicLocal Alignment Search Tool, J Mol Biol 215, 403-410). In addition, theinvention provides nucleic acids having a sequence about 60-70%,preferably about 70-80%, more preferably about 80-90%, more preferablyabout 90-95%, most preferably about 95-99% similar to SEQ ID NO: 1 asdetermined by Best Fit analysis using default settings.

[0020] The subject nucleic acids are of synthetic/non-natural sequencesand/or are recombinant, meaning they comprise a non-natural sequence ora natural sequence joined to nucleotide(s) other than that which it isjoined to on a natural chromosome. The subject recombinant nucleic acidscomprising the nucleotide sequence of disclosed vertebrate R5.24 nucleicacids, or fragments thereof, contain such sequence or fragment at aterminus, immediately flanked by (i.e. contiguous with) a sequence otherthan that which it is joined to on a natural chromosome, or flanked by anative flanking region fewer than 10 kb, preferably fewer than 2 kb,more preferably fewer than 500 bp, which is at a terminus or isimmediately flanked by a sequence other than that which it is joined toon a natural chromosome. While the nucleic acids are usually RNA or DNA,it is often advantageous to use nucleic acids comprising other bases ornucleotide analogs to provide modified stability, etc.

[0021] The subject nucleic acids find a wide variety of applicationsincluding use as translatable transcripts, hybridization probes, PCRprimers, diagnostic nucleic acids, etc.; use in detecting the presenceof R5.24 genes and gene transcripts and in detecting or amplifyingnucleic acids encoding additional R5.24 homologs and structural analogs.

EXAMPLES

[0022] We have developed a general method for expression cloning novel Gprotein coupled receptors as a strategy for identifying vertebrateodorant receptors. The sensitivity and flexibility of this techniqueallows the activation of multiple G protein pathways to be detected,even when the relevant receptor mRNA constitutes as little as 0.1% ofthe injected RNA population. Thus, this system facilitates thefunctional identification of cDNAs corresponding to any G proteincoupled receptor for which specific agonists are available. By using ourexpression cloning approach, we isolated from the goldfish olfactoryepithelium a cDNA encoding a receptor that is activated by amino acidodorants. Characterization of this receptor, receptor 5.24, reveals thatit is preferentially activated by arginine and lysine and interacts withthese compounds with high affinity (Kd=˜100 nM). Other amino acids bindto receptor 5.24 with lower affinity; parameters affecting bindingspecificity appear to include the structure and/or charge of the sidechain's terminal functional moiety, as well as its backbone length. Thereceptor demonstrates stereospecificity and does not appear to bindamino acid neurotransmitters found in the peripheral olfactory system.The observed affinity of this cloned receptor agrees well with the invivo threshold sensitivities of the goldfish olfactory system toarginine (ca. 1 nM). However, the cloned goldfish receptor appears to bedifferent from basic amino acid binding sites characterized in isolatedfish olfactory cilia, which show 50˜100-fold lower affinities for ligand(e.g., Cagan and Zeiger, 1978).

[0023] It has been suggested that amino acid odorant stimuli aretransduced by phospholipase-mediated pathways in fish (Huque and Bruch,1986; Restrepo et al., 1993). Consistent with these observations, ourresults demonstrate that odorant activation of the cloned goldfish aminoacid receptor leads to increased PI turnover in Xenopus oocytes as wellas in mammalian cells. No coupling to Gαs-like pathways is observed eventhough these these G protein subunits are present in both heterologouscell systems, indicating that the goldfish amino acid odorant receptorand all olfactory CaSR receptors, stimulate PI turnover in vivo.Interestingly, these receptors are expressed in microvillous neurons(see also Cao et al., 1998), which morphologically resemble the sensoryneurons of the VNO. Both the VNO as well as fish olfactory microvillousneurons do not appear to express cyclic nucleotide-gated channel alphasubunits (Berghard et al., 1996), which are required for transducingodorant-evoked cAMP elevations into changes in membrane potential inmammalian ciliated olfactory neurons (Brunet et al., 1996).

[0024] Receptor 5.24 shares sequence similarity to previously identifiedG protein receptors, including the CaSR, mGluR, and V2R families(Nalanishi et al., 1990; Hebert and Brown, 1995; Herrada and Dulac,1997; Matsunami and Buck, 1997; Ryba and Tirindelli, 1997). AdditionalCaSR-like receptors are also expressed in the goldfish olfactoryepithelium. Our results indicate that receptor 5.24 is a member of amultigene family of receptors expressed by olfactory sensory neurons,and together with our biochemical characterization of this receptorprovide direct evidence that the family of olfactory CaSR-like receptorsare in fact odorant receptors.

[0025] The mammalian V2R receptors have been proposed to constitute afamily of pheromone receptors based on their expression in the VNO(Herrada and Dulac, 1997; Matsunami and Buck, 1997; Ryba and Tirindelli,1997). It should be noted, however, that the VNO—a specialization of theolfactory apparatus in terrestrial vertebrates—receives both pheromonalas well as non-pheromonal cues (Halpern, 1987). While the ligandspecificities of the mammalian V2R receptors remain to be demonstrated,our data clearly show that at least one member of the goldfish receptorfamily, receptor 5.24, is an odorant receptor that recognizes a specificsubset of amino acid stimuli. Since amino acid odorants are notpheromones, the family of olfactory CaSR-like receptors, including themammalian V2R receptors, may in fact function to receive a wide varietyof stimuli that includes both pheromonal and non-pheromonal odorants. Wedisclose that receptors related to receptor 5.24 are used by the fish todetect other amino acid odorants.

[0026] Electrophysiological recordings from isolated salmon olfactoryneurons have demonstrated that ˜60% of the cells are sensitive to0.01-10 μM L-serine (Nevitt and Dittman, 1999). Similarly, single-unitrecordings from the catfish olfactory epithelium have shown that ˜40% ofthe olfactory neurons respond to 100 μM L-arginine (Kang and Caprio,1995). Multi-unit recordings from the goldfish olfactory epithelium,where activity from 5 cells was detected at each recording site, alsosuggest that a large fraction of olfactory neurons respond to arginine(25 out of 28 locations with spontaneous activity responded 100 μML-arginine), but few appeared sensitive to pheromones (e.g., only 25 outof 65 locations responded to 0.1 μM 15-ketoprostaglandin F2a). Thus, thewidespread expression of receptor 5.24 mRNA in goldfish olfactoryneurons is consistent with electrophysiological recordings whichindependently suggest that a large fraction of fish olfactory neuronsexpress amino acid odorant receptors.

[0027] The following descriptions of particular embodiments and examplesare offered by way of illustration and not by way of limitation. Unlesscontraindicated or noted otherwise, in these descriptions and throughoutthis specification, the terms “a” and “an” mean one or more, the term“or” means and/or and polynucleotide sequences are understood toencompass opposite strands as well as alternative backbones describedherein.

[0028] General Strategy for Expression Cloning of Odorant Receptors. Weelected to utilize the goldfish olfactory system, owing to the extensivephysiological and behavioral characterization of its responses to bothpheromonal and non-pheromonal olfactory stimuli in this species(Sorensen and Caprio, 1998; Sorensen et al., 1998). Our approach wasdesigned to allow for the detection of receptor activation of multiple Gprotein-mediated pathways—whereas previous studies have demonstratedthat odorant-evoked excitatory signaling in mammalian olfactory neuronsis mediated exclusively by the intracellular second messenger, cAMP(Brunet et al., 1996), in vitro biochemical studies have suggested thatstimulation of phosphatidyl inositol (PI) turnover, resulting in theproduction of the second messengers diacylglycerol and inositol1,4,5-trisphosphate (IP3), may mediate olfactory signaling in fish(Huque and Bruch, 1986; Restrepo et al., 1993). Thus, for expressioncloning of fish odorant receptors it seemed prudent to utilize a systemcapable of detecting activation of multiple signaling pathways.

[0029] The Xenopus oocyte provides a powerful method for expressioncloning certain G protein-coupled receptors, owing to the ability todetect increases in PI turnover through the IP3-mediated release ofCa2+from internal stores and the subsequent activation of Ca2+-dependentC1-channels (Masu et al., 1987). This cell does not normally exhibit anelectrophysiologic response to the activation of Gas (and thereforeadenylyl cyclase), however. We therefore engineered the oocyte toprovide a robust read-out for this G protein-dependent pathway (Lim etal., 1995). This method relies upon the ectopic expression of Gaolf (aGas-like isoform highly enriched in olfactory cilia; Jones and Reed,1989) and G protein-gated inwardly rectifying potassium channels(GIRKs), together with candidate receptors (Lim et al., 1995). Potassiumcurrents can be observed in response to gating of GIRK channels by freeG protein bg subunits following their dissociation from activatedGas-like or Gai subunits (Reuveny et al., 1994; Lim et al., 1995). Todetermine whether this system would be amenable to expression cloning,where a cDNA encoding the receptor of interest comprises only a smallfraction of a pool of cDNAs, we injected into oocytes RNA encoding thedopamine D1 receptor together with Gaolf and GIRK RNAs, with the amountof receptor RNA diluted 1,000-fold prior to injection. We couldtypically elicit robust agonist-dependent currents inreceptor-expressing oocytes. These controls indicated that a pool of˜1,000 cDNA clones containing a single receptor cDNA could still giverise to a detectable signal when expressed and activated in our assaysystem. Thus, oocytes expressing Gaolf and GIRK provide a means ofexpression cloning G protein-coupled receptors whose downstream couplingpathways are ambiguous.

[0030] Identification by Expression Cloning of a cDNA Encoding aGoldfish Odorant Receptor. RNA was synthesized from pools of goldfisholfactory cDNA clones (900 individual clones per pool) and injected intoXenopus oocytes together with synthetic RNAs encoding GIRK and Gaolf.Oocytes were then screened for responses upon exposure to odorantcocktails containing amino acids, bile acids, or sex pheromones.Odorants were tested at concentrations 100- to 1,000-fold higher thanthose required to elicit half-maximal physiological responses in vivo(Caprio, 1978; Sorensen et al., 1987; Sorensen et al., 1988; Michel andLubomudrov, 1995; see Experimental Procedures); these concentrations didnot elicit activity in oocytes injected only with GIRK and G proteinRNAs. Oocytes injected with RNA from one pool, pool 19, demonstrated arobust response to amino acids, but not to bile acids or sex pheromones.The response to amino acids was biphasic, beginning with an oscillatinginward current above the basal inward K+ current, followed by a declinein inward current to below the basal level. Such a biphasic effect isthought to be caused by Gaq-mediated activation of phospholipase C(responsible for the intial inward current via IP3) and protein kinase C(leading to suppression of the K+ current through phosphorylation ofGIRK channel subunits) (Sharon et al., 1997). Indeed, the oscillatinginward currents in response to amino acids were still observed inoocytes expressing pool 19 RNA without Gaolf or GIRK RNAs, suggestingthat the receptor contained in this pool interacts with aphospholipase-mediated pathway.

[0031] Iterative subdivision of pool 19 by sib-selection allowed theisolation of a single clone encoding a receptor, designated receptor5.24. Receptor 5.24 responds best to basic L-amino acids, showingroughly equivalent evoked currents upon activation by arginine andlysine, smaller responses to neutral aliphatic L-amino acids (e.g.,methionine, isoleucine, threonine, serine, alanine) and little or noresponse to acidic and aromatic L-amino acids (e.g., glutamate,aspartate, tyrosine, phenylalanine, tryptophan, histidine); the receptoris not activated by D-amino acids (all amino acids referred to hereafterare L-isomers unless stated otherwise).

[0032] High-Affinity Binding of Radiolabeled L-Arginine to the ClonedGoldfish Odorant Receptor. To determine the affinity of the clonedgoldfish amino acid receptor for basic amino acids, we characterized theligand-receptor interaction by radiolabeled ligand binding to receptorsexpressed in mammalian cells. Human embryonic kidney (HEK) 293 cellswere transfected with expression plasmids containing the receptor 5.24cDNA insert. Membranes prepared from receptor 5.24-expressing cellsexhibit saturable binding at concentrations of up to 1 mM 3H-arginine,and the extent of ligand binding is significantly higher than withmembranes from control cells. Further analysis of specific bindingactivity from multiple experiments indicates a receptor with a singlebinding site (Hill coefficient=0.95±0.07 [mean±SEM], n=4 determinations)with a dissociation constant (Kd) of 121±33 nM arginine (mean±SEM, n=4;range: 52-207 nM). We next wished to determine what second messengerpathway receptor 5.24 couples to in HEK 293 cells. Control cells orcells expressing receptor 5.24 were exposed to varying concentrations ofarginine and assayed for the accumulation of IP3 and cAMP. Arginineelicits a specific increase in IP3 in receptor 5.24-expressing cells ina dose-dependent manner. By way of contrast, arginine at 0.1 mM or 10 mMdoes not cause a detectable change in cAMP levels in these cells, eventhough activation of b-adrenergic receptors (expressed endogenously bythe host cell line) leads to increased cAMP accumulation. These resultsindicate that, as in Xenopus oocytes, receptor 5.24 preferentiallystimulates PI turnover in HEK 293 cells.

[0033] Structure-Activity Properties of Compounds Interacting with theCloned Goldfish Amino Acid Odorant Receptor. An understanding of howodorant receptors are used to encode olfactory information requires acharacterization of the odorant specificities of individual receptortypes. We therefore wished to determine the relative specificity ofreceptor 5.24 for structurally related ligands. Since this receptorbinds to arginine with high affinity, we screened other compounds forreceptor 5.24 binding by using a 3H-arginine displacement assay. Whilethese assays do not give information regarding whether a compoundfunctions as an agonist, partial agonist, or antagonist, they dononetheless allow insight into the molecular specificity of thereceptor. Briefly, 3H-arginine binding to membranes from receptor5.24-expressing HEK 293 cells was assayed in the absence or presence ofvarying concentrations of competitor ligands. Consistent with theirprofiles of receptor 5.24 activation in Xenopus oocytes, arginine andlysine displace 3H-arginine binding with similar concentrationdependencies, showing half-maximal inhibition (IC50) at ˜0.3 and 0.5 mM,respectively (corresponding to inhibition constants or Ki's of 80 and 90nM; see Table 3). Glutamate, which does not appear to activate receptor5.24 expressed in Xenopus oocytes, displaces 3H-arginine approximately80-fold less well than either unlabeled arginine or lysine (IC50=˜20 mM;Ki=6.7 mM). Interestingly, agmatine, a decarboxylated analogue ofarginine, displaces 3H-arginine very poorly (Ki>1 mM; see below andTable 4). TABLE 3 Binding Affinity of the Receptor for Amino Acids,Amino Acid Derivatives and Neurotransmitters. Binding affinity (Ki) wasdetermined by the ability of the individual amino acids to displace3H-L-arginine binding from membranes prepared from HEK 293 cellsexpressing receptor 5.24. AMINO ACID CLASS AMINO ACID Ki (μM) Basic sidechain L-Arginine 0.08 L-Lysine 0.09 Sulfur-containing side chainL-Cysteine 0.53 L-Methionine 0.81 Amide side chain L-Glutamine 0.32L-Asparagine 2.1 Acidic side chain L-Glutamate 6.7 L-Aspartate 27 Longaliphatic side chain L-Isoleucine 2.2 L-Leucine 4.4 L-Valine 6.2 Shortaliphatic side chain L-Serine 2.8 L-Threonine 3.2 L-Glycine 3.9L-Alanine 5.6 L-Proline 58 Aromatic side chain L-Tryptophan 4.1L-Phenylalanine 5.8 L-Histidine 13 L-Tyrosine 16 Arginine/Lysinederivatives Agmatine >1000 L-Citruline 0.96 L-Ornithine 1.00L-Homoarginine 1.63 L-NAME 1.02 Cadaverine >1000 Putrescine >1000Neurotransmitters γ-Aminobutyric Acid (GABA) >1000 Taurine >1000Carnosine >1000

[0034] TABLE 4 Odorant Cocktails Used for Screening the GoldfishOlfactory cDNA Library in Xenopus Ooctyes Amino Acids/Bile Acids L-AminoAcids Serine, Alanine, Methionine, Glutamic (Final concentration 50 mM)Acid, Arginine, Glutamine, Lysine, Histidine Bile Acids TaurocholicAcid, Taurolithocholic Acid (Final concentration 1 mM) Sulfate,Taurodeoxycholic Acid, Taurochenodeoxycholic Acid, Glycocholic Acid,Prostaglandins/Sex Steroids Prostaglandins Prostaglandin F2a,15-Ketoprostaglandin (Final concentration 100 mM) F2a Sex Steroids 17,20b-dihydroxy-4-pregnen-3-one, 17,20 (Final concentration 10 mM)b-dihydroxy-4-pregnen-3-one Sulfate

[0035] We performed comparative binding studies for 20 naturallyoccurring amino acids as well as amino acid analogues andneurotransmitters (Tables 3 and 4). A number of trends are evident fromthis analysis. First, receptor 5.24 apparently is tuned to recognizeamino acids containing basic R group side chains; of the 20 amino acidstested, arginine and lysine display the highest affinities. In addition,10- to >100-fold lower affinities are observed with arginine and lysineanalogues, and no specific interactions could be detected for the aminoacid neurotransmitters g-amino butyric acid (GABA), camosine, or taurine(Ki>1 mM), which are present in the peripheral olfactory system (Nicoll,1971; Collins, 1974; Margolis, 1974). Second, the side chain's terminalfunctional group is an important parameter in determining specificity.Substitution of the basic side chain with R groups containing amide(glutamine, asparagine), sulfur-containing (cysteine, methionine), orcarbamyl (citrulline) moieties results in a ˜4- to ˜25-fold decrease inaffinity. Amino acids with side chains containing terminal amide orsulfur-containing groups in general demonstrate higher affinity thanthose with aliphatic side chains lacking these structures. Substitutionwith acidic side chains (glutamate, aspartate) results in a large (˜80-to ˜300-fold) decrease in affinity. Amino acids containing cyclized(proline) or aromatic (tryptophan, phenylalanine, histidine, tyrosine)side groups in general interact with receptor 5.24 with low affinities.Third, specificity is based in part on the R group's carbon backbonelength, as illustrated by comparing lysine (Ki=0.09 mM) vs. omithine(Ki=1.0 mM; backbone shorter than lysine's by one carbon) and arginine(Ki=0.08 mM) vs. homoarginine (Ki=1.6 mM; backbone longer thanarginine's by one carbon). Tuning of this receptor based on carbon chainlength appears to be sharper than has been observed for a cloned ratodorant receptor, which shows a somewhat broad response profile for 7-,8-, 9-, and 10-carbon n-aliphatic aldehydes (Zhao et al., 1997).Finally, the a-carboxylic acid moiety is critical for receptor binding,as agmatine and cadaverine (decarboxylated analogues of arginine andlysine, respectively) show essentially no binding to receptor 5.24 (Ki's >1 mM, or greater than 10,000 times the Kd for arginine or lysine).However, the interaction with the carboxylic acid is probably notdependent on the negative charge per se, since the carboxy-methylatedarginine analogue, L-NAME, binds the receptor with modest affinity(Ki=1.0 mM).

[0036] The Goldfish Amino Acid Odorant Receptor Belongs to a Family ofCaSR-Like Receptors. The sequence of the goldfish receptor 5.24 cDNApredicts a protein with seven membrane spanning regions preceded by a566 amino acid N-terminal extracellular domain. Receptor 5.24 exhibitssignificant similarity to previously identified G protein-coupledreceptors, including the CaSR (Hebert and Brown, 1995), the family ofmGluR receptors (Tanabe et al., 1992), the family of vomeronasal V2Rreceptors (Herrada and Dulac, 1997; Matsunami and Buck, 1997; Ryba andTirindelli, 1997), a family of olfactory CaSR- and V2R-related receptorsfound in the puffer fish, fugu (Naito et al., 1998) and goldfish (Cao etal., 1998), and two putative taste receptors found in the mouse (Hoon etal., 1999). Receptor 5.24 shares between 25 and 33% amino acid sequenceidentity with these receptors, showing a somewhat greater degree ofsimilarity with human and fugu CaSR sequences. In spite of this weakhomology to the CaSR and mGluR sequences, receptor 5.24 is not activatedby calcium or glutamate.

[0037] The N-terminal extracellular domain of the mGluR is required forglutamate binding (O'Hara et al., 1993; Takahashi et al., 1993), andthis region of the metabotropic as well as ionotropic glutamatereceptors shows significant sequence similarity with bacterialperiplasmic amino acid binding proteins (Nakanishi et al., 1990; O'Haraet al., 1993). Molecular modeling of the mGluR1 N-terminal domain basedon the bacterial protein structures suggests that serine 165 andthreonine 188 may interact with glutamate by coordination of the aminoacid ligand's a-amino and a-carboxyl moieties; conservative mutations atthese positions in mGluR1 results in a significant reduction in agonistbinding (O'Hara et al., 1993). Interestingly, the corresponding tworesidues are conserved in receptor 5.24 (serine 152 and threonine 175),but not in the CaSR or in every V2R sequence. These residues in thegoldfish receptor probably serve to coordinate the high affinity bindingof amino acid odorants.

[0038] A Family of Olfactory CaSR-Like Receptors Related to the GoldfishAmino Acid Odorant Receptor. Recent work by others has identifiedmembers of a family of CaSR-like receptors that show similarity toreceptor 5.24 and are specifically expressed in the goldfish olfactoryepithelium (Cao et al., 1998). To identify additional receptor sequencesin this gene family, we performed PCR on goldfish olfactory cDNA usingdegenerate primers based on motifs conserved among the N-terminalregions of receptor 5.24, CaSR, mGluR, and V2R sequences. Subcloning andDNA sequencing of the resulting PCR products revealed numerous CaSR-likesequences (SEQ ID NOS:3-6) that can be grouped into 5 distinctsubfamilies. Within this portion of the N-terminal domain, the goldfishCaSR-like olfactory receptor subfamilies exhibit between 20 and 43%amino acid identity. Receptor 5.24 is the most divergent member of thegroup of receptors identified thus far, showing 20 to 27% similaritywith the other sequences in this region. The receptor 5.24 full lengthcDNA detects 1-2 bands in genomic DNA blots, suggesting that this geneexists as a single copy in the goldfish genome.

[0039] In addition, full-length CaSR-like sequences are readily isolatedfrom cDNA libraries using the foregoing techniques. For example, full,native length CaSR-like protein sequences SEQ ID NOS:7-8, 10 and 12 areencoded by full length goldfish cDNAs. Natural coding sequences for SEQID NOS:10 and 12 are shown as SEQ ID NOS:9 and 11, respectively.Furthermore, heterologous CaSR-like sequences are readily isolated usingthese techniques. For example, a zebrafish protein shown to befunctionally and structurally similar to goldfish 5.24 (ca. 70% aminoacid identity)is shown as SEQ ID NO: 14 (the natural coding sequence isshown as SEQ ID NO:13).

[0040] Expression Patterns of Goldfish Olfactory CaSR-Like Receptors.Analysis of receptor 5.24 expression by RNA blots revealed that the mRNAencoding this receptor is expressed in olfactory epithelium but not inbrain, kidney, liver, muscle, ovary, intestine, or testis. The receptor5.24 probe also detects at high stringency an MRNA in skin from thetrunk, gill, lips, tongue, and palatal organ. Similar RNA blot analysiswith probes for receptor 5.3 and receptor 3.13 indicate that these genesare expressed exclusively in olfactory epithelium.

[0041] RNA in situ hybridizations were performed to determine theexpression patterns of the goldfish olfactory CaSR-like sequences in theolfactory epithelium. We probed tissue sections with an 35S-labeledantisense RNA probe corresponding to the N-terminal extracellular domainof receptor 5.24. This is the most divergent region of this class ofreceptor, and therefore is expected to anneal only to receptor 5.24under the stringent conditions of hybridization used in theseexperiments. We found receptor 5.24 MRNA expressed widely over theapical and medial portions of the olfactory sensory epithelium-regionswhich contain the olfactory sensory neurons. In situ hybridizationsusing a digoxigenin-labeled probe confirm that receptor 5.24 isexpressed in a large fraction of cells in the neuronal layers of thesensory epithelium. These observations are consistent withelectrophysiological recordings which suggest that roughly half of theolfactory neurons in fish can respond to amino acid stimuli (Kang andCaprio, 1995; Nevitt and Dittman, 1999). In situ hybridizations using aprobe for receptor 5.3 indicate that this MRNA is also localized to alarge subset of cells. In contrast to the broad patterns of receptor5.24 and receptor 5.3 expression, other CaSR-like receptors areexpressed in punctate patterns within the olfactory epithelium. Probesfor receptor 3.13, receptor 9, and receptor 10 subfamily membershybridize to a small subset of cells (ca. 1˜5% each).

[0042] We noticed that signal strengths for receptor 5.24 in situhybridizations were consistently weaker than for the other receptors.This appears to be a peculiarity of the receptor 5.24 MRNA and not dueto a low level of its expression, as screening of the goldfish olfactorycDNA library with olfactory CaSR-like sequences reveals that these RNAsare expressed at roughly equivalent levels (each sequence is representedat between 1 in ˜100,000 clones [receptors 5.24 and 5.3] to 1 in 600,000clones [receptor 10.8]).

[0043] Cells expressing the goldfish CaSR-like receptors localize moreapically than we typically observe for olfactory neurons expressing theolfactory cyclic nucleotide-gated ion channel (Goulding et al., 1992;see below) or odorant receptors belonging to the family originallydescribed by Buck and Axel (1991). The fish olfactory epitheliumcontains two major classes of sensory cells, the ciliated andmicrovillous neurons, that are segregated along the apical-basal axis(Yamamoto, 1982). The microvillous cells reside in the apical portion ofthe epithelium in a zone above and distinct from the ciliated neurons,whose cell bodies lie medially. Previous in situ hybridization studieshave shown that the class of receptors originally described by Buck andAxel (1991), as well as the cyclic nucleotide-gated ion channel, areexpressed in the medially-disposed ciliated olfactory neurons in fish(Goulding et al., 1992; Ngai et al., 1993a; Ngai et al., 1993b). Thus,the goldfish CaSR-like receptors are probably expressed in microvillousolfactory neurons (see also Cao et al., 1998).

[0044] Localization of Receptor 5.24 mRNA Expression in Non-OlfactoryTissue. The expression of receptor 5.24 mRNA in external epitheliaraises the question as to whether this receptor might be playing achemosensory function outside of the olfactory system. These epitheliacontain both taste buds as well as solitary chemosensory cells; both ofthese systems are sensitive to amino acid stimuli in fish (Sorensen andCaprio, 1998), although facial nerve recordings in goldfish indicatethat arginine is a poor taste stimulus in this species. We thereforeperformed additional in situ hybridizations to determine whetherreceptor 5.24 is indeed expressed in these chemosensory systems.Exemplary data showed representative tissue sections of gill rakers thatwere hybridized with a digoxigenin-labeled receptor 5.24 probe. Therakers are non-respiratory structures associated with the gill archesand are covered with an epithelium containing taste buds and solitarychemosensory cells. Examination of these tissue sections as well asnumerous others similarly hybridized with the receptor probe indicatesthat this sequence is expressed widely in the overlying epithelium, butis distinctly excluded from taste buds. In addition, the epithelialcells expressing receptor 5.24 are far too numerous to be accounted forsolely by the solitary chemosensory cells, which are relatively rare anddispersed in the epithelium (hence the term “solitary;” Sorensen andCaprio, 1998). Thus, these results argue against a role for receptor5.24—an odorant receptor tuned to recognize basic amino acids —innon-olfactory chemosensory transduction.

[0045] Experimental Procedures: Expression Cloning. Poly(A)+RNA wasprepared from adult male goldfish olfactory rosettes. CDNA wassynthesized using an oligo(dT) primer and double stranded DNA wasligated directionally into pSPORT-1 plasmid (Life Technologies, Inc.)via 5′ Sal 1 and 3′ Not I restriction sites. Ligation reactions wereintroduced into E. coli by electroporation. Plasmid DNA was preparedfrom pools of 900-1000 clones, linearized with Not I, purified, and usedas template for in vitro transcription with T7 RNA polymerase. Forproduction of cRNAs encoding G protein and GIRK subunits, cDNAs forGaolf (Jones and Reed, 1989) and the GIRK subunits Kir 3.1 (Reuveny etal., 1994) and Kir 3.4 (Ashford et al., 1994) were amplified by PCRusing Pfu polymerase and subcloned into the RNA expression vector,pGEMHE (Liman et al., 1992). Following in vitro transcriptions, cRNAswere precipitated in LiCl and resuspended in water.

[0046] Oocytes were removed from anesthetized Xenopus laevis and treatedwith collagenase. Forty nanograms of cRNA from each cDNA library pool(˜40 pg cRNA/clone) was injected per oocyte, together with cRNAsencoding Gaolf and the GIRK subunits Kir 3.1 and Kir 3.4 (˜30 pg each).Approximately 30 pools were assayed before these primary screens werehalted. Injected oocytes were incubated at 17° C. for over 80 hoursprior to electrophysiological recordings. Recordings were performed bytwo-electrode voltage clamping using an Axoclamp-2A amplifier (AxonInstruments) or a Dagan Calif.-1 amplifier (Dagan Corp.). Dataacquisition and analysis were performed using pCLAMP software (AxonInstruments). Membrane potential was held at −80 mV. For trialsinvolving GIRK, oocytes were first perfused with Na-MBSH (88 mM NaCl, 1mM KCl, 2.4 MM NaHCO3, 10 mM Hepes, pH 7.5, 0.82 mM MgSO4[7H20], 0.33 mMCa[NO3]2[4H20], 0.41 mM CaCl2[2H20]), and then switched into K-MBSH,which contains elevated K+(88 mM KCl, 1 mM NaCl) until the basal currenthad stabilized (˜45 seconds) before challenging with agonist. Recordingson oocytes injected with receptor 5.24 in the absence of Gaolf and/orGIRK subunits were performed in Na-MBSH. Oocytes were exposed tosolutions containing different agonists by switching bathing solutionswith an 8 channel valve (Hamilton).

[0047] DNA sequencing was carried out with a Pharmacia AlfExpresssequencer. Sequences were analyzed using MacVector software.

[0048] Mammalian Cell Culture and DNA Transfections. For celltransfections, the receptor 5.24 cDNA insert was subcloned into twoexpression vectors: CMVI, which utilizes a human cytomegalovirus (CMV)immediate early promoter-enhancer plus the CMV intron A sequence todrive expression, and 608RX-2.2L, which is similar to CMVI except thecDNA insert is followed by an internal ribosome entry site(IRES)—enhanced green fluorescent protein (EGFP) coding sequence;608RX-2.2L also contains a puromycin resistance gene. For transientassays, HEK 293 cells expressing the SV40 large T antigen (293 TSAcells) were transfected with the CMVI-receptor 5.24 or CMVI controlplasmid using lipofectamine (Life Technologies) and harvested at 48hours for membrane preparations. For stable cell lines, 293-TSA cellswere transfected with the 608RX-2.2L-receptor 5.24 plasmid or a control608RX-2.2L plasmid and selected in puromycin. Colonies showing highlevels of EGFP fluorescence (as judged by epifluorescence microscopy)were picked, expanded, and screened for 3H-L-arginine binding (seebelow). In five independent receptor 5.24-transfected stable cell lines,receptor densities (Rt) varied between 0.5 and 6.0 pmol L-argininebinding sites/mg membrane protein. In four independent608RX-2.2L-transfected control cell lines, EGFP expression wascomparable to receptor 5.24-transfected cells, but L-arginine bindingwas indistinguishable from untransfected cells (Rt=0.01-0.03 pmol/mg).All competition binding assays and signal transduction studies wereperformed using the stable cell line 5.24-20 (Rt=2.0 pmol/mg) and acontrol cell line (2.2-9) stably transfected with vector alone (Rt=0.03pmol/mg).

[0049] Membrane Preparations and Ligand Binding Assays. Membranes forligand binding assays were prepared by washing confluent cells threetimes with phosphate buffered saline (PBS), detaching cells with aPBS-based enzyme-free dissociation solution, and resuspending cells inice-cold 5.0 mM Hepes, pH 7.4, 1.0 mM EDTA, 1.0 μg/ml leupeptin, 0.5 mMPMSF. All subsequent manipulations were performed on ice. After a 30 minincubation, the cell suspension was homogenized and centrifuged at100,000×g for 30 min. Cell membrane pellets were washed twice byresuspension and centrifugation in binding buffer (20 mM Hepes, pH 7.4,1.0 mM EDTA, 2.0 mM MgCl2, 1.0 μg/ml leupeptin, 0.5 mM PMSF),resuspended in binding buffer, and frozen at −80° C.

[0050] Saturation binding assays were performed using 20-25 μg ofmembrane protein in a final volume of 100 μl binding buffer andincreasing concentrations of [2,3-3H] L-arginine (specific activity=40Ci/mmol; Dupont NEN). Non-specific binding was measured in the presenceof 500 μM unlabeled L-arginine. Competition assays were performed with500 nM [2,3-3H] L-arginine and increasing concentrations of competitorligands. All incubations were performed at 4° C. for 60 min andterminated by rapid filtration through Whatman GF/C filters pretreatedwith ice cold 0.1% polyethyleneimine. Filters were washed three timeswith 4.0 ml ice cold 20 mM Hepes, pH 7.4, and retained radioactivity wasmeasured by scintillation counting. All experiments were performed intriplicate and repeated at least twice. Data were analyzed by nonlinearcurve fitting using Origin software (Microcal). The concentration ofcompetitor which caused 50% inhibition of 3H-L-arginine binding (IC50)was determined by non-linear curve-fitting; inhibition constants werecalculated according to the equation Ki=(IC50)/(1+[3H-arginine]/Kd).

[0051] IP3 and cAMP Measurements. For measurements of IP3 and cAMP, celllines 5.24-20 and 2.2-9 (negative control) were plated at densities of3.5×105 cells/well in 24 well plates and incubated overnight in DMEMcontaining 10% FBS. All subsequent manipulations prior to reactiontermination were performed at 37° C. in 5% CO2. Prior to odorantexposure, confluent cells were washed twice with PBS to remove freeamino acids and incubated for 3 hours in amino acid-free media (Earle'sBalance Salt Solution containing 0.1% BSA) to allow for recovery frompotential desensitization of receptors or downstream signaling pathways.For IP3 assays, cells were washed once with PBS, pre-incubated for 15min in PBS containing 10 mM LiCl, and then exposed to odorants in 400 μlPBS/10 mM LiCl for 30 min. IP3 levels were determined by a radioreceptorcompetition assay (Dupont NEN). For cAMP measurements, after the 3 houramino acid-free preincubation cells were washed once with PBS,preincubated for 15 min in PBS containing 0.5 mM IBMX, and exposed toodorants in 400 μl PBS/0.5 mM IBMX for 30 min. cAMP levels weredetermined by radioimmunoassay (DuPont NEN).

[0052] PCR. PCR was performed to identify additional CaSR-like odorantreceptor cDNAs. Three degenerate oligonucleotide primers were designedbased on an alignment of receptor 5.24, mGluR (Duvoisin et al., 1995),human CaSR (Garrett et al., 1995), and mammalian V2R2 (Herrada andDulac, 1997; Matsunami and Buck, 1997; Ryba and Tirindelli, 1997)sequences:

[0053] Primer A: corresponding to amino acids 211-215 in receptor 5.24;

[0054] Primer B: corresponding to amino acids 518-514 in receptor 5.24;

[0055] Primer C: corresponding to amino acids 755-751 in receptor 5.24.

[0056] Nested PCR was performed on plasmid library pools containingapproximately 20,000 clones. DNA from each library pool was used astemplate for a primary PCR reaction using a 5′T7 primer with Primer C.Primary PCR reactions were separated on a 1% agarose gel and productsbetween 1-4 kb were excised, eluted, and used as template for asecondary PCR reaction using Primer A and Primer B. Secondary PCRproducts were electrophoresed on a 1% agarose gel and fragments of ˜1 kbwere subcloned into the TA plasmid vector (Invitrogen) and sequenced.

[0057] RNA Blot Analysis and In Situ Hybridizations. The distribution ofreceptor 5.24 mRNA in goldfish tissues was determined by RNA blotanalysis, using 32P-labeled antisense RNAs as probes at high stringency(Ambion). One-half microgram of poly(A)-enriched RNA from each goldfishtissue analyzed was electrophoresed under denaturing conditions, blottedto a nylon membrane, and probed with a 600 nt RNA probe corresponding toamino acids 389-600 of receptor 5.24. Since the full-length receptor5.24 cDNA appears to recognize a single gene in genomic DNA blots, andsequences encoding this region comprise the most divergent portion ofthis class of receptors, this probe most likely is entirely specific forreceptor 5.24 RNA under stringent hybridization conditions. As acontrol, the membrane was subsequently hybridized to a goldfish b-actinRNA probe.

[0058] RNA in situ hybridizations were performed on 20 mm-thick freshfrozen tissue sections from adult goldfish olfactory rosettes,essentially as described (Barth et al., 1996; Barth et al., 1997).Slides were hybridized with 35S-labeled (107 cpm/ml) ordigoxigenin-labeled (1 mg/ml) probes at 60-65° C. for a minimum of 16hours and washed in 0.2×SSC at 65° C. Slides hybridized with 35S probeswere additionally treated with 20 mg/ml RNase A, rewashed in 0.2×SSC at65° C., dehydrated, dipped in Kodak NTB-2 emulsion, exposed for 14-28days at 4° C., developed, and counterstained with toluidine blue.Digoxigenin-labeled probes were visualized with an alkalinephosphatase-conjugated anti-digoxigenin antibody and chromogenicdevelopment in NBT/BCIP.

[0059] For receptor 5.24 localization, a ˜1.8 kb Pst I fragmentcorresponding to the first 600 amino acids of the full-length receptorwas subcloned into pBluescript and used for in vitro transcription of35S- and digoxigenin labeled RNA probes. Digoxigenin-labeled probes forreceptors 3.13 and 5.3 were synthesized from the cloned −1 kb PCRinserts derived from these receptors' N-terminal domains. To identifymembers of the receptor family initially identified by Buck and Axel(1991), degenerate reverse transcription PCR was carried out on goldfisholfactory RNA, as described previously (Barth et al., 1996). Onegoldfish clone from this class of receptors, designated D1/113-6, wasused to synthesize digoxigenin-labeled RNA probes. Cells expressing theolfactory cyclic nucleotide-gated channel were localized with adigoxigenin-labeled probe synthesized from a ˜2.5 kb full-lengthzebrafish cDNA (see Barth et al., 1996).

REFERENCES

[0060] Abe, K., et al. (1993). J. Biol. Chem. 268, 12,033-12,039.

[0061] Ashford, M. L., Bond, C. T., Blair, T. A., and Adelman, J. P.(1994). Nature 370, 456-459.

[0062] Barth, A. L., Dugas, J. C., and Ngai, J. (1997). Neuron 19,359-369.

[0063] Barth, A. L., Justice, N. J., and Ngai, J. (1996). Neuron 16,23-34.

[0064] Berghard, A., and Buck, L. B. (1996). J. Neurosci. 16, 909-918.

[0065] Berghard, A., Buck, L. B., and Liman, E. R. (1996). Proc. Natl.Acad. Sci. USA 93, 2365-2369.

[0066] Brunet, L. J., Gold, G. H., and Ngai, J. (1996). Neuron 17,681-693.

[0067] Buck, L., and Axel, R. (1991). Cell 65, 175-187.

[0068] Buck, L. B. (1996). Ann. Rev. Neurosci. 19, 517-544.

[0069] Cagan, R. H., and Zeiger, W. N. (1978). Proc. Natl. Acad. Sci.USA 75, 4679-4683.

[0070] Cao, Y., Oh, B. C., and Stryer, L. (1998). Proc. Natl. Acad. Sci.USA 95, 11,987-11,992.

[0071] Caprio, J. (1978). J. Comp. Physiol. 123, 357-371.

[0072] Caprio, J., and Byrd, R. P. (1984). J. Gen. Physiol. 84, 403-422.

[0073] Collins, G. G. (1974). Brain Res. 76, 447-59.

[0074] Dreyer, W. J. (1998). Proc. Natl. Acad. Sci. USA 95, 9072-9077.

[0075] Dulac, C., and Axel, R. (1995). Cell 83, 195-206.

[0076] Duvoisin, R. M., Zhang, C., and Ramonell, K. (1995). J. Neurosci.15, 3075-3083.

[0077] Friedrich, R. W., and Korsching, S. I. (1997). Neuron 18,737-752.

[0078] Garrett, J. E., et al. (1995). J. Biol. Chem. 270, 12919-12925.

[0079] Goulding, E. H., et al. (1992). Neuron 8, 45-58.

[0080] Halpem, M. (1987). Annu.Rev.Neurosci.10,325-362.

[0081] Hara, T. J. (1994). Acta Physiol. Scand. 152, 207-217.

[0082] Hebert, S. C., and Brown, E. M. (1995). Curr. Opin. Cell Biol. 7,484-492.

[0083] Herrada, G., and Dulac, C. (1997). Cell 90, 763-773.

[0084] Hoon, M. A., et al. (1999). Cell 96, 541-551.

[0085] Huque, T., and Bruch, R. C. (1986). Biochem. Biophys. Res. Comm.137, 36-42.

[0086] Jia, C., and Halpern, M. (1996). Brain Res. 719, 117-128.

[0087] Jones, D. T., and Reed, R. R. (1989). Science 244, 790-795.

[0088] Kang, J., and Caprio, J. (1995). J. Neurophysiol. 73, 172-177.

[0089] Krautwurst, D., Yau, K. W., and Reed, R. R. (1998). Cell 95,917-926.

[0090] Lim, N. F., et al. (1995). J. Gen. Physiol. 105, 421-439.

[0091] Liman, E. R., Tytgat, J., and Hess, P. (1992). Neuron 9, 861-871.

[0092] Malnic, B., Hirono, J., Sato, T., and Buck, L. B. (1999) Cell 96,713-723.

[0093] Margolis, F. L. (1974). Science 184,909-911.

[0094] Masu, M., et al. (1991). Nature 349, 760-765.

[0095] Masu, Y., et al. (1987). Nature 329, 836-838.

[0096] Matsunami, H., and Buck, L. B. (1997). Cell 90, 775-784.

[0097] Medler, K. F., Hansen, A., and Bruch, R. C. (1998). Neuroreport9, 4103-4107.

[0098] Michel, W. C., and Derbidge, D. S. (1997). Brain Res. 764,179-87.

[0099] Michel, W. C., and Lubomudrov, L. M. (1995). J. Comp. Physiol. A177, 191-199.

[0100] Naito, T., et al. (1998). Proc. Natl. Acad. Sci. USA 95,5178-5181.

[0101] Nakanishi, N., Shneider, N. A., and Axel, R. (1990). Neuron 5,569-581.

[0102] Nef, P., et al. (1992). Proc. Natl. Acad. Sci. USA 89, 8948-8952.

[0103] Nef, S., and Nef, P. (1997). Proc. Natl. Acad. Sci. USA 94,4766-4771.

[0104] Nevitt, G., and Dittman, A. (1999). Integr. Biol. 1, in press.

[0105] Ngai, J., et al. (1993a). epithelium. Cell 72, 667-680.

[0106] Ngai, J., Dowling, M. M., Buck, L., Axel, R., and Chess, A.(1993b). Cell 72, 657-666.

[0107] Nicoll, R. A. (1971). Brain Res. 35, 137-49.

[0108] O'Hara, P. J., et al. (1993). Neuron 11, 41-52.

[0109] Parmentier, M., et al. (1992). Nature 355, 453-455.

[0110] Ressler, K. J., Sullivan, S. L., and Buck, L. B. (1993). Cell 73,597-609.

[0111] Restrepo, D., Boekhoff, I., and Breer, H. (1993). Amer. J.Physiol. 264, 906-911.

[0112] Reuveny, E., et al (1994). Nature 370, 143-146.

[0113] Ryba, N. J., and Tirindelli, R. (1997). Neuron 19, 371-379.

[0114] Sengupta, P., Chou, J. C., and Bargmann, C. I. (1996). Cell 84,899-909.

[0115] Sharon, D., Vorobiov, D., and Dascal, N. (1997). J. Gen. Physiol.109, 477-490.

[0116] Shepherd, G. M. (1994). Neuron 13, 771-790.

[0117] Sorensen, P. W., and Caprio, J. C. (1998). Chemoreception. In ThePhysiology of Fishes, 2nd edition, D. H. Evans, ed. (Boca Raton: CRCPress), pp. 375-405.

[0118] Sorensen, P. W., et al. (1998). Curr. Opin. Neurobiol. 8,458-467.

[0119] Sorensen, P. W., Hara, T. J., and Stacey, N. E. (1987). J. Comp.Physiol. A 160, 305-313.

[0120] Sorensen, P. W., et al. (1988). Biol. Reprod. 39, 1039-1050.

[0121] Takahashi, K., et al. (1993). J. Biol. Chem. 268, 19,341-19,345.

[0122] Tanabe, Y., Masu, M., Ishii, T., Shigemoto, R., and Nakanishi, S.(1992). Neuron 8, 169-79.

[0123] Yamamoto, M. (1982). In Chemoreception in Fishes, T. J. Hara, ed.(Amsterdam: Elsevier Scientific Publishing Company), pp. 39-59.

[0124] Zhang, Y., et al. (1997). Proc. Natl. Acad. Sci. USA 94,12,162-12,167.

[0125] Zhao, H., et al. (1997). Science 279, 237-242.

[0126] Zhou, Q. Y., et al. (1990). Nature 347, 76-80.

[0127] All publications and patent applications cited in thisspecification and all references cited therein are herein incorporatedby reference as if each individual publication or patent application orreference were specifically and individually indicated to beincorporated by reference. Although the foregoing invention has beendescribed in some detail by way of illustration and example for purposesof clarity of understanding, it will be readily apparent to those ofordinary skill in the art in light of the teachings of this inventionthat certain changes and modifications may be made thereto withoutdeparting from the spirit or scope of the appended claims.

1 14 1 2877 DNA Carassius auratus CDS (34)..(2664) 1 gtcgacccacgcgtccgaca gcctaaagca gtg atg gct ggt ttg gat ttg agc 54 Met Ala Gly LeuAsp Leu Ser 1 5 ctg gta ctc atg ttg tct gtg ctg gca gga gtc aga gag gtttca ctg 102 Leu Val Leu Met Leu Ser Val Leu Ala Gly Val Arg Glu Val SerLeu 10 15 20 aca cag gtt aac caa caa gga gtc ata gcc cct gga gac atc attatt 150 Thr Gln Val Asn Gln Gln Gly Val Ile Ala Pro Gly Asp Ile Ile Ile25 30 35 gga ggt ctt ttt ccc atc cat gag gca gcg gag gca gtg aac ttc act198 Gly Gly Leu Phe Pro Ile His Glu Ala Ala Glu Ala Val Asn Phe Thr 4045 50 55 ggc tta aac agc ttc tct tct ttt cag cat cca gtc tgc aac aga tac246 Gly Leu Asn Ser Phe Ser Ser Phe Gln His Pro Val Cys Asn Arg Tyr 6065 70 tac aca aaa ggt cta aat cag gct cta gct atg att cat gct gtg gaa294 Tyr Thr Lys Gly Leu Asn Gln Ala Leu Ala Met Ile His Ala Val Glu 7580 85 atg gca aac caa tcc ccc atg ttg agc agt ttg aat tta act ctt gga342 Met Ala Asn Gln Ser Pro Met Leu Ser Ser Leu Asn Leu Thr Leu Gly 9095 100 tat cgc atc tat gac aca tgt tct gat gtc acg act gca ctt tgg gcc390 Tyr Arg Ile Tyr Asp Thr Cys Ser Asp Val Thr Thr Ala Leu Trp Ala 105110 115 gtc caa gat ctc aca cgg ccg tac tcc tac tgt gac tca caa act aac438 Val Gln Asp Leu Thr Arg Pro Tyr Ser Tyr Cys Asp Ser Gln Thr Asn 120125 130 135 tct tct caa cct gtc cag cca ata atg gca gta att ggg ccc tcttct 486 Ser Ser Gln Pro Val Gln Pro Ile Met Ala Val Ile Gly Pro Ser Ser140 145 150 tct gag atc tcc atc gca gtt gcc agg gaa ctc aac ctt ctg atgatt 534 Ser Glu Ile Ser Ile Ala Val Ala Arg Glu Leu Asn Leu Leu Met Ile155 160 165 cca cag ata agt tat gca tct aca gct acg att ctt agt gac aaaagt 582 Pro Gln Ile Ser Tyr Ala Ser Thr Ala Thr Ile Leu Ser Asp Lys Ser170 175 180 cgt ttt cct gct ttc atg agg act gtc cca aat gat gag tac caaacc 630 Arg Phe Pro Ala Phe Met Arg Thr Val Pro Asn Asp Glu Tyr Gln Thr185 190 195 cat gcc atg gta caa ctt ctg aag gac aat aaa tgg acc tgg gttggg 678 His Ala Met Val Gln Leu Leu Lys Asp Asn Lys Trp Thr Trp Val Gly200 205 210 215 att atc att aca gat gga gac tat ggg cgt tct gcc atg gaaagt ttt 726 Ile Ile Ile Thr Asp Gly Asp Tyr Gly Arg Ser Ala Met Glu SerPhe 220 225 230 gtt aag cac act gaa agg gag gga att tgt gtg gcc ttt aaggtg atc 774 Val Lys His Thr Glu Arg Glu Gly Ile Cys Val Ala Phe Lys ValIle 235 240 245 cta cca gat tca cta gca gac gaa caa aaa tta aac atc cacatc aac 822 Leu Pro Asp Ser Leu Ala Asp Glu Gln Lys Leu Asn Ile His IleAsn 250 255 260 gag act gtg gac atc att gaa aaa aat act aag gtt aat gtggtg gtc 870 Glu Thr Val Asp Ile Ile Glu Lys Asn Thr Lys Val Asn Val ValVal 265 270 275 tca ttt gct aag tca tct caa atg aag ttg cta tat gag ggcctg cgt 918 Ser Phe Ala Lys Ser Ser Gln Met Lys Leu Leu Tyr Glu Gly LeuArg 280 285 290 295 agt agg aac gtt cca aaa aat aaa gta tgg gtg gcc agcgat aac tgg 966 Ser Arg Asn Val Pro Lys Asn Lys Val Trp Val Ala Ser AspAsn Trp 300 305 310 tct acc tct aaa aat att cta aaa gac gta aac ctc tcagat atc gga 1014 Ser Thr Ser Lys Asn Ile Leu Lys Asp Val Asn Leu Ser AspIle Gly 315 320 325 aat ata ctg ggc ttc acc ttc aag agt gga aat gtt acagct ttt ctt 1062 Asn Ile Leu Gly Phe Thr Phe Lys Ser Gly Asn Val Thr AlaPhe Leu 330 335 340 caa tac ctt aag gat ctg aag ttt gga agt gaa gct aagatg aac aat 1110 Gln Tyr Leu Lys Asp Leu Lys Phe Gly Ser Glu Ala Lys MetAsn Asn 345 350 355 tca ttc ttg gaa gaa ttt tta aaa ctg cct gaa ata ggaaat gct gca 1158 Ser Phe Leu Glu Glu Phe Leu Lys Leu Pro Glu Ile Gly AsnAla Ala 360 365 370 375 aac gct gta cag gaa cag att aaa aac aca cat ttggac atg gtc ttc 1206 Asn Ala Val Gln Glu Gln Ile Lys Asn Thr His Leu AspMet Val Phe 380 385 390 agt gtt cag atg gca gtc agt gct att gct aaa gctgtg gtt gaa cta 1254 Ser Val Gln Met Ala Val Ser Ala Ile Ala Lys Ala ValVal Glu Leu 395 400 405 tgt gta gaa aga caa tgc aag acc cct tca gct atccaa ccc tgg gag 1302 Cys Val Glu Arg Gln Cys Lys Thr Pro Ser Ala Ile GlnPro Trp Glu 410 415 420 ctc tta aaa cag ctg agg aac gtc act ttt gag aaagaa gga gtc atg 1350 Leu Leu Lys Gln Leu Arg Asn Val Thr Phe Glu Lys GluGly Val Met 425 430 435 tac aat ttt gac gcc aat gga gac att aat ttg ggctat gat gtc tgc 1398 Tyr Asn Phe Asp Ala Asn Gly Asp Ile Asn Leu Gly TyrAsp Val Cys 440 445 450 455 cta tgg gat gac gat gaa tct gaa aaa aat gacata ata gca gaa tat 1446 Leu Trp Asp Asp Asp Glu Ser Glu Lys Asn Asp IleIle Ala Glu Tyr 460 465 470 tat cca tct aac agc agt ttc act ttt aca aggaag aat cta agt aat 1494 Tyr Pro Ser Asn Ser Ser Phe Thr Phe Thr Arg LysAsn Leu Ser Asn 475 480 485 att gag aat gtg tta tct aag tgt tcg gac agctgt caa cca ggg gag 1542 Ile Glu Asn Val Leu Ser Lys Cys Ser Asp Ser CysGln Pro Gly Glu 490 495 500 tac aaa aaa aca gca gag ggt cag cac act tgctgt tat gag tgt ctt 1590 Tyr Lys Lys Thr Ala Glu Gly Gln His Thr Cys CysTyr Glu Cys Leu 505 510 515 gcc tgc gcc gaa aac caa tac tcc aac cac acagat gca gac aca tgt 1638 Ala Cys Ala Glu Asn Gln Tyr Ser Asn His Thr AspAla Asp Thr Cys 520 525 530 535 tct aag tgc gac act gag agc ttg tgg tcaaac gct aat agc tca aaa 1686 Ser Lys Cys Asp Thr Glu Ser Leu Trp Ser AsnAla Asn Ser Ser Lys 540 545 550 tgt tat ccc aag ttt tat gag tac ttt gagtgg aat agt ggt ttt gcc 1734 Cys Tyr Pro Lys Phe Tyr Glu Tyr Phe Glu TrpAsn Ser Gly Phe Ala 555 560 565 atc gcc ctg ctg acg ctg gct gcc ctc ggcatc cta ctc ctc atc tca 1782 Ile Ala Leu Leu Thr Leu Ala Ala Leu Gly IleLeu Leu Leu Ile Ser 570 575 580 atg tcc gca ctg ttc ttc tgg caa agg aactct cta gtg gtt aaa gct 1830 Met Ser Ala Leu Phe Phe Trp Gln Arg Asn SerLeu Val Val Lys Ala 585 590 595 gca ggt gga cca ctt tgt cat ctg atc cttttc tcc ctg ctg ggc agt 1878 Ala Gly Gly Pro Leu Cys His Leu Ile Leu PheSer Leu Leu Gly Ser 600 605 610 615 ttt atc agt gtc att ttc ttt gtg ggtgaa ccg agc aat gag tcc tgt 1926 Phe Ile Ser Val Ile Phe Phe Val Gly GluPro Ser Asn Glu Ser Cys 620 625 630 agg gta agg cag gtc atc ttt ggc ctgagc ttc acg ctg tgt gtt tca 1974 Arg Val Arg Gln Val Ile Phe Gly Leu SerPhe Thr Leu Cys Val Ser 635 640 645 tgc atc tta gtg aag tcc ttg aag atcctt ctg gcg ttc cag atg aac 2022 Cys Ile Leu Val Lys Ser Leu Lys Ile LeuLeu Ala Phe Gln Met Asn 650 655 660 cta gag ctg aag gag ctt ctt cgt aaactc tac aag ccg tat gtg atc 2070 Leu Glu Leu Lys Glu Leu Leu Arg Lys LeuTyr Lys Pro Tyr Val Ile 665 670 675 gtt tgc atg tgt atg ggg ctt cag gtcacc att tgc act ctt tgg ctg 2118 Val Cys Met Cys Met Gly Leu Gln Val ThrIle Cys Thr Leu Trp Leu 680 685 690 695 acc ttg cac agg cct ttt att gaaaaa gtg gtg caa ccc aaa tcc att 2166 Thr Leu His Arg Pro Phe Ile Glu LysVal Val Gln Pro Lys Ser Ile 700 705 710 ctc ctg gaa tgc aat gag ggt tcagat ttg atg ttt ggg tta atg ctg 2214 Leu Leu Glu Cys Asn Glu Gly Ser AspLeu Met Phe Gly Leu Met Leu 715 720 725 ggt tac ata gtt ttg ctg gcg ctgata tgt ttc act ttt gct tat aaa 2262 Gly Tyr Ile Val Leu Leu Ala Leu IleCys Phe Thr Phe Ala Tyr Lys 730 735 740 ggc agg aaa ctt ccg cag aag tataac gaa gca aag ttc atc aca ttt 2310 Gly Arg Lys Leu Pro Gln Lys Tyr AsnGlu Ala Lys Phe Ile Thr Phe 745 750 755 ggt atg ctc atc tac ctc atg gcctgg gtc att ttt atc cca gtg cac 2358 Gly Met Leu Ile Tyr Leu Met Ala TrpVal Ile Phe Ile Pro Val His 760 765 770 775 gtg acc acc agt ggc aaa tatgta ccg gct gtg gag gta gtt gtt att 2406 Val Thr Thr Ser Gly Lys Tyr ValPro Ala Val Glu Val Val Val Ile 780 785 790 ctc att tca aac tat ggg atcctg agc tgc cac ttt ttg cca aaa tgt 2454 Leu Ile Ser Asn Tyr Gly Ile LeuSer Cys His Phe Leu Pro Lys Cys 795 800 805 tac ata att att ttt aaa aaggag tat aat acc aaa gat gca ttc ttg 2502 Tyr Ile Ile Ile Phe Lys Lys GluTyr Asn Thr Lys Asp Ala Phe Leu 810 815 820 aaa aat gtt ttt gaa tac gccaga aag agc tct gag aac atc agg ggc 2550 Lys Asn Val Phe Glu Tyr Ala ArgLys Ser Ser Glu Asn Ile Arg Gly 825 830 835 ttg tct gga act gat cca cacagt aaa act gac aat tca gtc tat gtc 2598 Leu Ser Gly Thr Asp Pro His SerLys Thr Asp Asn Ser Val Tyr Val 840 845 850 855 ata tcc aac ccg tca cttgtg cct gag gag aaa caa gtt tct gta cca 2646 Ile Ser Asn Pro Ser Leu ValPro Glu Glu Lys Gln Val Ser Val Pro 860 865 870 gaa ata gac aat gtg ctttaaagtagtt gcaagaattt gagatcacga 2694 Glu Ile Asp Asn Val Leu 875gtcaaagcaa ccattcagac aaaatttggt cttcatttga catgaaactt gtatttcaca 2754taatgatctt taaaaatacc aaacttcatg atgatcattt taaattatga atactttcat 2814ttgtggaaaa caaataaaat gtgtatattt gtgtatattt gaaattaaaa aaaaaaaaaa 2874aaa 2877 2 877 PRT Carassius auratus 2 Met Ala Gly Leu Asp Leu Ser LeuVal Leu Met Leu Ser Val Leu Ala 1 5 10 15 Gly Val Arg Glu Val Ser LeuThr Gln Val Asn Gln Gln Gly Val Ile 20 25 30 Ala Pro Gly Asp Ile Ile IleGly Gly Leu Phe Pro Ile His Glu Ala 35 40 45 Ala Glu Ala Val Asn Phe ThrGly Leu Asn Ser Phe Ser Ser Phe Gln 50 55 60 His Pro Val Cys Asn Arg TyrTyr Thr Lys Gly Leu Asn Gln Ala Leu 65 70 75 80 Ala Met Ile His Ala ValGlu Met Ala Asn Gln Ser Pro Met Leu Ser 85 90 95 Ser Leu Asn Leu Thr LeuGly Tyr Arg Ile Tyr Asp Thr Cys Ser Asp 100 105 110 Val Thr Thr Ala LeuTrp Ala Val Gln Asp Leu Thr Arg Pro Tyr Ser 115 120 125 Tyr Cys Asp SerGln Thr Asn Ser Ser Gln Pro Val Gln Pro Ile Met 130 135 140 Ala Val IleGly Pro Ser Ser Ser Glu Ile Ser Ile Ala Val Ala Arg 145 150 155 160 GluLeu Asn Leu Leu Met Ile Pro Gln Ile Ser Tyr Ala Ser Thr Ala 165 170 175Thr Ile Leu Ser Asp Lys Ser Arg Phe Pro Ala Phe Met Arg Thr Val 180 185190 Pro Asn Asp Glu Tyr Gln Thr His Ala Met Val Gln Leu Leu Lys Asp 195200 205 Asn Lys Trp Thr Trp Val Gly Ile Ile Ile Thr Asp Gly Asp Tyr Gly210 215 220 Arg Ser Ala Met Glu Ser Phe Val Lys His Thr Glu Arg Glu GlyIle 225 230 235 240 Cys Val Ala Phe Lys Val Ile Leu Pro Asp Ser Leu AlaAsp Glu Gln 245 250 255 Lys Leu Asn Ile His Ile Asn Glu Thr Val Asp IleIle Glu Lys Asn 260 265 270 Thr Lys Val Asn Val Val Val Ser Phe Ala LysSer Ser Gln Met Lys 275 280 285 Leu Leu Tyr Glu Gly Leu Arg Ser Arg AsnVal Pro Lys Asn Lys Val 290 295 300 Trp Val Ala Ser Asp Asn Trp Ser ThrSer Lys Asn Ile Leu Lys Asp 305 310 315 320 Val Asn Leu Ser Asp Ile GlyAsn Ile Leu Gly Phe Thr Phe Lys Ser 325 330 335 Gly Asn Val Thr Ala PheLeu Gln Tyr Leu Lys Asp Leu Lys Phe Gly 340 345 350 Ser Glu Ala Lys MetAsn Asn Ser Phe Leu Glu Glu Phe Leu Lys Leu 355 360 365 Pro Glu Ile GlyAsn Ala Ala Asn Ala Val Gln Glu Gln Ile Lys Asn 370 375 380 Thr His LeuAsp Met Val Phe Ser Val Gln Met Ala Val Ser Ala Ile 385 390 395 400 AlaLys Ala Val Val Glu Leu Cys Val Glu Arg Gln Cys Lys Thr Pro 405 410 415Ser Ala Ile Gln Pro Trp Glu Leu Leu Lys Gln Leu Arg Asn Val Thr 420 425430 Phe Glu Lys Glu Gly Val Met Tyr Asn Phe Asp Ala Asn Gly Asp Ile 435440 445 Asn Leu Gly Tyr Asp Val Cys Leu Trp Asp Asp Asp Glu Ser Glu Lys450 455 460 Asn Asp Ile Ile Ala Glu Tyr Tyr Pro Ser Asn Ser Ser Phe ThrPhe 465 470 475 480 Thr Arg Lys Asn Leu Ser Asn Ile Glu Asn Val Leu SerLys Cys Ser 485 490 495 Asp Ser Cys Gln Pro Gly Glu Tyr Lys Lys Thr AlaGlu Gly Gln His 500 505 510 Thr Cys Cys Tyr Glu Cys Leu Ala Cys Ala GluAsn Gln Tyr Ser Asn 515 520 525 His Thr Asp Ala Asp Thr Cys Ser Lys CysAsp Thr Glu Ser Leu Trp 530 535 540 Ser Asn Ala Asn Ser Ser Lys Cys TyrPro Lys Phe Tyr Glu Tyr Phe 545 550 555 560 Glu Trp Asn Ser Gly Phe AlaIle Ala Leu Leu Thr Leu Ala Ala Leu 565 570 575 Gly Ile Leu Leu Leu IleSer Met Ser Ala Leu Phe Phe Trp Gln Arg 580 585 590 Asn Ser Leu Val ValLys Ala Ala Gly Gly Pro Leu Cys His Leu Ile 595 600 605 Leu Phe Ser LeuLeu Gly Ser Phe Ile Ser Val Ile Phe Phe Val Gly 610 615 620 Glu Pro SerAsn Glu Ser Cys Arg Val Arg Gln Val Ile Phe Gly Leu 625 630 635 640 SerPhe Thr Leu Cys Val Ser Cys Ile Leu Val Lys Ser Leu Lys Ile 645 650 655Leu Leu Ala Phe Gln Met Asn Leu Glu Leu Lys Glu Leu Leu Arg Lys 660 665670 Leu Tyr Lys Pro Tyr Val Ile Val Cys Met Cys Met Gly Leu Gln Val 675680 685 Thr Ile Cys Thr Leu Trp Leu Thr Leu His Arg Pro Phe Ile Glu Lys690 695 700 Val Val Gln Pro Lys Ser Ile Leu Leu Glu Cys Asn Glu Gly SerAsp 705 710 715 720 Leu Met Phe Gly Leu Met Leu Gly Tyr Ile Val Leu LeuAla Leu Ile 725 730 735 Cys Phe Thr Phe Ala Tyr Lys Gly Arg Lys Leu ProGln Lys Tyr Asn 740 745 750 Glu Ala Lys Phe Ile Thr Phe Gly Met Leu IleTyr Leu Met Ala Trp 755 760 765 Val Ile Phe Ile Pro Val His Val Thr ThrSer Gly Lys Tyr Val Pro 770 775 780 Ala Val Glu Val Val Val Ile Leu IleSer Asn Tyr Gly Ile Leu Ser 785 790 795 800 Cys His Phe Leu Pro Lys CysTyr Ile Ile Ile Phe Lys Lys Glu Tyr 805 810 815 Asn Thr Lys Asp Ala PheLeu Lys Asn Val Phe Glu Tyr Ala Arg Lys 820 825 830 Ser Ser Glu Asn IleArg Gly Leu Ser Gly Thr Asp Pro His Ser Lys 835 840 845 Thr Asp Asn SerVal Tyr Val Ile Ser Asn Pro Ser Leu Val Pro Glu 850 855 860 Glu Lys GlnVal Ser Val Pro Glu Ile Asp Asn Val Leu 865 870 875 3 315 PRT Carassiusauratus 3 Thr Val Arg Ser Arg Asn Asp Tyr Gly Asn Asn Gly Ile Ala AlaPhe 1 5 10 15 Glu Glu Ala Ala Lys Glu Glu Gly Val Cys Ile Glu Tyr SerGlu Ala 20 25 30 Ile Leu Asn Asn Asp Pro Gln Glu Gln Phe Leu Lys Thr LeuGlu Val 35 40 45 Ile Lys Lys Gly Thr Ala Arg Val Val Leu Ala Phe Ile AlaLeu Gly 50 55 60 Asp Phe Leu Pro Leu Leu Lys Val Ile Leu Gln His Asn IleThr Gly 65 70 75 80 Ile Gln Trp Val Gly Ser Glu Ser Trp Ile Thr Ser GlnThr Leu Ala 85 90 95 Glu Thr Lys Glu Tyr Ser Phe Leu Ser Gly Ala Val GlyPhe Ala Ile 100 105 110 Ala Asn Ala Lys Ile Met Gly Leu Arg Glu Phe LeuVal Asn Val His 115 120 125 Pro Tyr Lys Glu Pro Lys Asn Glu Leu Leu LysGlu Phe Trp Glu Ile 130 135 140 Val Phe Gln Cys Ser Phe Asn Ser Ile GlySer Gly Cys Thr Gly Ser 145 150 155 160 Glu Arg Leu Ala Glu Leu Gln AsnGlu Tyr Thr Asp Val Ser Glu Leu 165 170 175 Arg Ile Ala Asn Lys Val TyrThr Ala Val Tyr Ala Ile Ala Tyr Thr 180 185 190 Leu His Asn Ile Leu LysGlu Phe Arg Thr Ser Thr Asn Ser Ser Lys 195 200 205 Ile Gly Trp Pro IlePro Gln Met Val Leu Lys Tyr Met Arg Asp Val 210 215 220 Arg Phe Thr ValLys Thr Gly Glu Glu Ile Phe Phe Asp Glu Ser Gly 225 230 235 240 Asp ProVal Ala Arg Tyr Asp Leu Val Asn Trp Gln Ser Ala Glu Asp 245 250 255 GlySer Met Arg Phe Glu Leu Val Gly Leu Tyr Asp Ser Ser Leu Pro 260 265 270Ser Glu His Leu Gln Val Asn Gln Glu His Ile Leu Trp Ala Glu Lys 275 280285 Ser Gly Gln Leu Pro Val Ser Val Cys Ser Glu Ile Cys Pro Pro Gly 290295 300 Thr Arg Lys Ala Val Gln Lys Gly Arg Pro Val 305 310 315 4 323PRT Carassius auratus 4 Ala Leu Ser Asn Asp Asn Asp Tyr Gly Lys Asn GlyIle Ala Thr Phe 1 5 10 15 Ile Lys Ala Ala Gln Glu Glu Gly Val Cys IleGlu Tyr Ser Gln Ala 20 25 30 Phe Glu Ser Thr Gly Ser Lys Thr Ser Leu LysAsn Ile Val Asp Thr 35 40 45 Ile Arg Thr Ser Thr Ser Lys Val Ile Met AlaPhe Met Ser His Arg 50 55 60 Glu Ile Lys Ile Leu Val Asp Glu Leu Tyr ArgGln Asn Ile Thr Gly 65 70 75 80 Leu Gln Trp Ile Gly Ser Asp Ala Trp IleThr Asp Asp Ser Leu Ala 85 90 95 Asp Ser Gln Gly Asn Thr Leu Leu Ile GlySer Ile Gly Phe Thr Val 100 105 110 Arg Asn Ala Lys Ile Pro Gly Leu GlyPro Phe Leu Gln Lys Leu Asn 115 120 125 Pro Ser Gln Phe Pro Lys Ser MetPhe Leu Lys Glu Phe Trp Glu Ser 130 135 140 Ile Phe Gln Cys Ser Leu SerPro Asn Ala Leu Gln Arg Ala Cys Asn 145 150 155 160 Gly Ser Glu His LeuLys Tyr Val Lys His Pro Phe Thr Asp Val Ser 165 170 175 Asp Leu Arg TyrVal Asn Asn Val Tyr Asn Ala Val Tyr Ala Ile Ala 180 185 190 His Ala LeuHis Asn Leu Leu Ser Cys Asn His Gln Lys Gly Pro Phe 195 200 205 Ala AsnVal Thr Cys Ala Gln Pro Thr Ile Ile Gln Pro Trp Gln Ile 210 215 220 LeuHis Tyr Met Gln Thr Val Asn Phe Thr Met Asn Gly Gly Glu Ser 225 230 235240 Val Phe Phe Asp Ser Lys Gly Asp Ser Pro Ala Arg Tyr Glu Leu Val 245250 255 Asn Leu Gln Asn Ile Thr Lys Gly Thr Met Glu Val Val Thr Ile Gly260 265 270 Tyr Tyr Asp Ala Ile Gln Pro Arg Gly Gln Gln Phe Thr Met AsnAsn 275 280 285 Val Asn Ile Thr Trp Gly Gly Gly Leu Arg Thr Val Pro ValSer Val 290 295 300 Cys Ser Glu Ser Cys Pro Leu Gly Thr Arg Lys Ala ValGln Lys Gly 305 310 315 320 Arg Pro Ile 5 300 PRT Carassius auratus 5Ala Val Asn Ser Asp Asn Asp Tyr Gly Asn Asn Gly Met Ala Ile Phe 1 5 1015 Leu Lys Thr Ala Thr Glu Glu Gly Ile Cys Val Glu Tyr Ser Val Lys 20 2530 Phe Leu Arg Thr Glu His Glu Lys Ile Arg Asn Val Val Asp Ile Ile 35 4045 Lys Gln Gly Thr Thr Lys Val Ile Val Ala Phe Leu Thr Gly Phe Glu 50 5560 Met Lys Ser Leu Ile Glu Gln Leu Gly Ile Gln Asn Ile Thr Gly Leu 65 7075 80 Gln Met Ile Gly Val Glu Ala Trp Ile Thr Ser Lys Ser Leu Met Thr 8590 95 Pro Asn Ser Phe His Val Leu Gly Gly Ser Leu Gly Phe Ala Val Arg100 105 110 Lys Ile Gln Ile Glu Gly Phe Ala Asp Tyr Val Met Lys Ala PheTrp 115 120 125 Asp Thr Ala Phe Pro Cys Ser Phe Asn Ala Lys Leu Asn CysSer Arg 130 135 140 Tyr Gln Asp Leu Ser Val Val Lys Asn Tyr Asn Asp AspVal Pro Glu 145 150 155 160 Gln Arg Phe Leu Ser Tyr Val Tyr Lys Ala ValTyr Ala Val Ala His 165 170 175 Ser Leu His Ser Leu Leu Lys Cys Arg GluArg Asp Gly Cys Glu Glu 180 185 190 Gly Leu Thr Ile Gln Pro Gln Gln MetVal Glu Ala Leu Lys Lys Val 195 200 205 Asn Phe Thr Leu Lys Thr Gly AspHis Val Trp Phe Asp Ser Thr Gly 210 215 220 Gly Ala Val Ala Gln Tyr GluIle Val Asn Trp Gln Gln Asp Ser Asp 225 230 235 240 Gly Ser Phe Arg PheLys Ser Val Gly Tyr Tyr Asp Ala Ser Leu Pro 245 250 255 Pro Asp Gln ArgPhe Val Ile Ile Thr Lys Asn Ile Ile Trp Ala Arg 260 265 270 Gly Gln LeuGlu Lys Pro Arg Ser Val Cys Ser Glu Ser Cys Pro Pro 275 280 285 Gly ThrArg Lys Ala Ala Gln Lys Gly Arg Pro Val 290 295 300 6 323 PRT Carassiusauratus 6 Cys Ile Ala Ala Glu Asp Asp Tyr Gly Lys Tyr Gly Ile Lys ArgPhe 1 5 10 15 Lys Glu Val Val Glu Glu Ala Gly Val Cys Ile Ser Phe SerGlu Thr 20 25 30 Leu Pro Lys Val Ser Asn Pro Glu Ala Ile Glu Arg Ile ValGln Thr 35 40 45 Val Pro Asp Ser Thr Ala Lys Ile Ile Val Val Phe Ser SerAsp Val 50 55 60 Asp Met Ser Pro Leu Val Gly Glu Leu Leu Arg Asn Asn ValThr Asn 65 70 75 80 Arg Thr Trp Ile Ala Ser Glu Ala Trp Val Thr Ser AlaAla Ile Ser 85 90 95 Arg His Pro Asp Ile Leu Pro Val Leu Gly Gly Thr IleGly Phe Ala 100 105 110 Val Lys Arg Ala Glu Ile Pro Gly Leu Lys Glu HisLeu Leu Ser Val 115 120 125 Thr Pro Tyr Asn Asp Thr Leu Thr Glu Glu PheTrp Gly Ile Val Phe 130 135 140 Asn Cys Thr Leu Asn Tyr Arg Gln Val LeuArg Gly Thr Arg Arg Cys 145 150 155 160 Thr Gly Glu Glu Met Leu Glu LysLeu Asn Asn Thr Phe Thr Asp Val 165 170 175 Ser Gln Leu Arg Ile Thr TyrAsn Val Tyr Lys Ala Val Tyr Ala Val 180 185 190 Ala His Ala Leu His AsnLeu Glu His Cys Lys Pro Gly Ser Gly Pro 195 200 205 Phe Glu Asn Gly ThrCys Ala Asp Ile Thr Lys Phe Glu Pro Trp Gln 210 215 220 Leu Met Tyr TyrLeu Lys Asn Leu Arg Tyr Thr Val Pro His Thr Lys 225 230 235 240 Glu GluIle Tyr Phe His Asp Gly Asp Val Asp Gly Phe Tyr Glu Ile 245 250 255 LeuAsn Trp Gln Ser Asp Ser Glu Gly Gly Ile Ala Tyr Thr Pro Ile 260 265 270Gly Tyr Tyr Asn Ser Thr Ala Ala Pro Glu Glu Arg Leu Ile Ile Asn 275 280285 Asn Gly Ser Ile Ile Trp Asn Asn Asp Ile Leu Glu Thr Pro Arg Ser 290295 300 Val Cys Ser Glu Arg Cys Gln Pro Gly Thr Arg Met Gly Ile Arg Gln305 310 315 320 Gly Glu Pro 7 835 PRT Carassius auratus 7 Met Leu LeuPhe Leu Tyr Thr Leu Thr Leu Phe Asn His Phe His Thr 1 5 10 15 Lys AlaGlu Lys Ile Leu Cys Gln Met Met Gly Asp Ala Lys Tyr Pro 20 25 30 Leu LeuSer Lys Asp Gly Glu Ala Thr Ile Gly Gly Ile Phe Ala Met 35 40 45 His SerLys Glu Thr Leu Pro Ser Phe Glu Phe Thr Gln Lys Pro Gln 50 55 60 Pro LeuLeu Cys Ser Ser Val Asn Leu Pro Asp Phe Arg Leu Ala Gln 65 70 75 80 IleMet Ile Phe Ala Ile Glu Glu Ile Asn Arg Ser Gln Met Leu Leu 85 90 95 ProAsn Val Ser Ile Gly Tyr Gln Ile Tyr Asp Thr Cys Ser Ser Arg 100 105 110Met Ser Ser Met Ser Ala Thr Met Gly Leu Met Asn Gly Pro Glu Phe 115 120125 Ala Ala Gly Glu Thr Cys Asn Gly Glu Ser Ser Ile His Ala Ile Ile 130135 140 Gly Glu Thr Glu Ser Ser Ala Thr Val Ile Leu Ser Arg Thr Thr Gly145 150 155 160 Pro Phe Lys Ile Pro Val Ile Ser His Thr Ala Ser Cys GluCys Leu 165 170 175 Ser Asn Arg Lys Asp His Pro Ser Phe Phe Arg Thr IleSer Ser Asp 180 185 190 Tyr His Gln Ser Arg Ala Leu Ala Tyr Ile Val LysHis Leu Gly Trp 195 200 205 Ser Trp Val Gly Thr Val Asn Ser Asp Asn AspTyr Gly Asn Tyr Gly 210 215 220 Met Ala Ile Phe Leu Asn Thr Ala Gln LysGlu Gly Ile Cys Val Glu 225 230 235 240 Tyr Ser Glu Arg Phe Tyr Arg ThrGlu Pro Glu Lys Leu Lys Lys Val 245 250 255 Val Asp Thr Ile Lys Lys GlyThr Ala Lys Val Ile Val Ala Phe Val 260 265 270 Ser Phe Ile Glu Met GlyLeu Leu Ile Asp Gln Leu Asn Thr Leu Asn 275 280 285 Ile Thr Gly Leu GlnIle Ile Gly Val Glu Gly Trp Ile Thr Ser Lys 290 295 300 Ser Leu Ile ThrPro Lys Ser Phe Gln Val Met Gly Gly Ser Leu Gly 305 310 315 320 Phe AlaLeu Arg Lys Ile Asn Leu Glu Gly Phe Ser Asp Tyr Val Val 325 330 335 LysXaa Phe Trp Asp Thr Ala Phe Pro Cys Ser Gln Ile Lys Gly Asn 340 345 350Ile Ser Gln His Glu Ile Asn Cys Xaa Lys Tyr Gln Asp Leu Leu Ala 355 360365 Leu Lys Lys Tyr Asn Glu Asp Val Pro Glu Xaa Xaa Tyr Ser Ser His 370375 380 Val Tyr Lys Ala Val Tyr Ala Val Ala His Ser Leu His Ser Leu Leu385 390 395 400 Lys Cys Lys Glu Gln Xaa Gly Cys Glu Lys Asp Leu Thr IleGln Pro 405 410 415 Gln Gln Val Val Glu Ala Leu Lys Lys Val Asn Phe ThrVal Lys Phe 420 425 430 Gly Asp Arg Val Trp Phe Asp Arg Thr Gly Ala AlaVal Ala Gln Tyr 435 440 445 Glu Val Val Asn Trp Gln Gln Asp Ser Asp GlySer Leu His Phe Lys 450 455 460 Ser Val Gly Tyr Tyr Asp Ala Ser Leu ProPro Asp Gln Gln Phe Val 465 470 475 480 Leu Lys Thr Glu Asn Ile Ile TrpAla Lys Gly Gln Leu Glu Lys Pro 485 490 495 Asn Ser Val Cys Ser Glu SerCys Leu Pro Gly Thr Arg Lys Ala Ala 500 505 510 Gln Lys Gly Arg Pro ValCys Cys Tyr Asp Cys Ile Pro Cys Ala Glu 515 520 525 Gly Glu Ile Ser AsnGlu Thr Asp Ser Asn Asn Cys Lys Gln Cys Pro 530 535 540 Arg Glu Tyr TrpSer Asn Ala Glu Lys Thr Lys Cys Val Leu Lys Ala 545 550 555 560 Val GluPhe Leu Ser Phe Thr Glu Val Met Gly Ile Val Leu Ala Phe 565 570 575 PheSer Leu Phe Gly Ala Gly Leu Thr Ala Leu Val Ala Ile Leu Phe 580 585 590Tyr Arg Met Arg Asp Thr Pro Ile Val Lys Ala Asn Asn Ser Glu Leu 595 600605 Ser Phe Leu Leu Leu Phe Ser Leu Thr Leu Cys Phe Leu Cys Ser Leu 610615 620 Thr Phe Ile Gly Gln Pro Asn Glu Trp Ser Cys Met Leu Arg His Thr625 630 635 640 Ala Phe Gly Ile Thr Phe Val Leu Cys Ile Ser Cys Val LeuGly Lys 645 650 655 Thr Ile Val Val Leu Met Ala Phe Lys Ala Thr Leu ProGly Ser Asn 660 665 670 Val Met Lys Trp Phe Gly Pro Ala Gln Gln Arg LeuSer Val Leu Ala 675 680 685 Leu Thr Phe Ile Gln Ile Leu Ile Cys Val LeuTrp Leu Thr Ile Ser 690 695 700 Pro Pro Phe Pro Tyr Lys Asn Met Lys TyrPhe Lys Glu Lys Ile Ile 705 710 715 720 Leu Glu Cys Ser Leu Gly Ser SerIle Ser Phe Trp Ala Val Leu Gly 725 730 735 Tyr Ile Gly Leu Leu Ala ValLeu Cys Phe Ile Leu Ala Phe Leu Ala 740 745 750 Arg Thr Leu Pro Asp AsnPhe Asn Glu Ala Lys Phe Ile Thr Phe Ser 755 760 765 Met Leu Ile Phe CysAla Val Trp Ile Thr Phe Ile Pro Ala Tyr Val 770 775 780 Ser Ser Pro GlyLys Tyr Thr Val Ala Val Glu Ile Phe Ala Ile Leu 785 790 795 800 Ala SerSer Phe Gly Leu Leu Phe Cys Ile Phe Ala Pro Lys Cys Tyr 805 810 815 IleIle Leu Leu Lys Pro Asp Gln Asn Thr Lys Lys His Met Met Gly 820 825 830Lys Thr Phe 835 8 856 PRT Carassius auratus 8 Met Ala Lys Arg Thr IlePro Leu Val Leu Leu Leu Leu Val Val Tyr 1 5 10 15 Gly Val Cys Val ProAla Ser Ala Gln Val Cys Arg Leu Leu Gly Leu 20 25 30 Pro Ala Leu Pro LeuLeu Ser Ala His Lys Asp Ile Asn Ile Gly Ala 35 40 45 Ile Phe Ser Phe HisArg Ser Ala Leu Leu Lys Met His Pro Phe Thr 50 55 60 Ser Lys Pro Glu ProThr Thr Cys Ile Ser Phe Asn Leu Arg Glu Phe 65 70 75 80 Lys Phe Ala GlnThr Leu Ile Phe Ala Ile Glu Glu Ile Asn Asn Ser 85 90 95 Thr Gln Leu LeuPro Gly Val Ser Leu Gly Tyr Lys Ile Tyr Asp Ser 100 105 110 Cys Ser SerVal Ala Leu Thr Val Leu Ser Gly Met Ala Leu Met Asn 115 120 125 Gly TyrGlu Glu Thr Leu Ser Asp Thr Ser Cys Ser Arg Pro Pro Ala 130 135 140 ValHis Ala Ile Val Gly Glu Ser Asn Ser Ser Pro Thr Ile Gly Leu 145 150 155160 Ala Ser Leu Val Gly Pro Phe Ser Leu Pro Val Ile Ser His Phe Ala 165170 175 Thr Cys Ala Cys Leu Ser Asn Arg Lys Met Tyr Pro Ser Phe Phe Arg180 185 190 Thr Ile Pro Ser Asp Tyr Tyr Gln Ser Arg Ala Leu Ala Lys LeuVal 195 200 205 Lys His Phe Gly Trp Thr Trp Val Gly Thr Val Arg Ser ArgSer Asp 210 215 220 Tyr Gly Ser Asn Gly Ile Ala Ala Phe Glu Glu Ser AlaLys Glu Glu 225 230 235 240 Gly Ile Cys Ile Glu Tyr Ser Glu Ala Ile PheLys Thr Asp Pro Gln 245 250 255 Asp Gln Phe Leu Lys Thr Val Glu Val IleLys Lys Gly Thr Ala Arg 260 265 270 Val Val Leu Ala Phe Ile Ala Leu GlyAsp Phe Val Pro Leu Leu Lys 275 280 285 Val Ile Ser Gln His Asn Ile ThrGly Ile Gln Trp Val Gly Ser Glu 290 295 300 Ser Trp Ile Thr Ser Arg ThrLeu Ala Glu Thr Lys Glu Tyr Ser Phe 305 310 315 320 Leu Ser Gly Ala ValGly Phe Ala Ile Ala Asn Ala Lys Leu Met Gly 325 330 335 Leu Arg Glu PheLeu Val Asn Val His Pro Asp Gln Glu Pro Asn Asn 340 345 350 Glu Leu LeuLys Glu Phe Trp Glu Thr Thr Phe Gln Cys Ser Leu Ser 355 360 365 Asn SerGly Ser Gly Gly Cys Thr Gly Ser Glu Arg Ile Ala Glu Leu 370 375 380 GlnAsn Glu Tyr Thr Asp Val Ser Glu Leu Arg Ile Ala Asn Lys Val 385 390 395400 Tyr Thr Ala Val Tyr Ala Ile Ala Gln Thr Leu His Asn Ile Leu Lys 405410 415 Asp Ile Lys Ser Ser Thr Lys Ser Ser Lys Gly Glu Arg Pro Thr Pro420 425 430 Gln Lys Val Leu Glu Tyr Ile Gly Gly Val Lys Phe Thr Val LysThr 435 440 445 Gly Glu Glu Ile Phe Phe Asp Ala Ser Gly Asn Pro Val AlaArg Tyr 450 455 460 Asp Leu Val Asn Trp Gln Pro Val Gln Asp Gly Ser LeuGln Phe Lys 465 470 475 480 Asn Val Gly Phe Tyr Asp Ser Ser Leu Pro SerGlu Gln His Leu Gln 485 490 495 Val Asn Gln Glu His Ile Leu Trp Thr GlyAsp Ser Gly Gln Leu Pro 500 505 510 Val Ser Val Cys Ser Glu Thr Cys ProPro Gly Thr Arg Lys Ala Val 515 520 525 Gln Lys Gly Arg Pro Val Cys CysTyr Asp Cys Ile Pro Cys Gly Glu 530 535 540 Gly Glu Ile Ser Asn Gly ThrAsp Ser Asn Asp Cys Phe Pro Cys Asp 545 550 555 560 Leu Glu Tyr Trp SerAsn Glu Ser Asn Asp Arg Cys Val Leu Lys Val 565 570 575 Ile Glu Phe LeuSer Tyr Thr Glu Ile Met Gly Met Val Leu Cys Ile 580 585 590 Phe Ser PheIle Gly Val Leu Leu Thr Thr Ile Val Ser Phe Leu Phe 595 600 605 Tyr LeuHis Lys Glu Thr Pro Ile Val Arg Ala Asn Asn Ser Glu Leu 610 615 620 SerPhe Leu Leu Leu Phe Ser Leu Thr Leu Cys Phe Leu Cys Ser Leu 625 630 635640 Thr Phe Ile Gly Arg Pro Thr Glu Trp Ser Cys Met Leu Arg His Thr 645650 655 Ala Phe Gly Ile Thr Phe Val Leu Cys Ile Ser Cys Ile Leu Gly Lys660 665 670 Thr Ile Val Val Leu Met Ala Phe Lys Ala Thr Leu Pro Gly SerAsn 675 680 685 Val Met Lys Trp Phe Gly Pro Leu Gln Gln Gln Leu Ser ValVal Ser 690 695 700 Leu Thr Leu Ile Gln Met Ile Ile Cys Val Leu Trp LeuThr Ile Ser 705 710 715 720 Pro Pro Phe Pro Tyr Met Asn Leu Ser Tyr TyrArg Glu Lys Ile Ile 725 730 735 Leu Glu Cys Asn Val Gly Ser Asp Leu AlaPhe Trp Ala Val Leu Gly 740 745 750 Tyr Thr Gly Leu Leu Ser Ile Leu CysPhe Val Leu Ala Phe Leu Ala 755 760 765 Arg Lys Leu Pro Asp Asn Phe AsnGlu Ala Lys Phe Ile Thr Phe Ser 770 775 780 Met Leu Ile Phe Cys Ala ValTrp Leu Thr Phe Ile Pro Ala Tyr Val 785 790 795 800 Ser Ser Pro Gly LysPhe Thr Val Ala Val Glu Ile Phe Ala Ile Leu 805 810 815 Ala Ser Ser PheSer Leu Leu Phe Cys Ile Phe Ala Pro Lys Cys Tyr 820 825 830 Ile Ile LeuLeu Lys Pro Glu Lys Xaa His Lys Glu Thr Asn Asp Gly 835 840 845 Xaa LysHis Met Gln Ser Leu Trp 850 855 9 2923 DNA Carassius auratus CDS(39)..(2600) 9 cccgggtcga cccacgcgtc cgcatacata ctactggt atg gca aag agcact gtg 56 Met Ala Lys Ser Thr Val 1 5 tca ctg ctt cta ctg ctg gtg gtggtg cat ggt gtc ttt gtg cca gct 104 Ser Leu Leu Leu Leu Leu Val Val ValHis Gly Val Phe Val Pro Ala 10 15 20 tca gca caa ctc tgc agt ctg ctt ggtcac tct gca ttt cct gta ctt 152 Ser Ala Gln Leu Cys Ser Leu Leu Gly HisSer Ala Phe Pro Val Leu 25 30 35 tct gca gaa aga gac atc aac att gga gcaatt ttc tca att cac aga 200 Ser Ala Glu Arg Asp Ile Asn Ile Gly Ala IlePhe Ser Ile His Arg 40 45 50 agt gct ctg cta aag atg cac cct ttc act tccaaa cca gag cca aca 248 Ser Ala Leu Leu Lys Met His Pro Phe Thr Ser LysPro Glu Pro Thr 55 60 65 70 aca tgc ctc agg tta aac ttg cgt gaa ttt aaattt gct cag aca ttt 296 Thr Cys Leu Arg Leu Asn Leu Arg Glu Phe Lys PheAla Gln Thr Phe 75 80 85 att ttt gcc att gag gag ata aat aac agc aca cagctt ttg cct gga 344 Ile Phe Ala Ile Glu Glu Ile Asn Asn Ser Thr Gln LeuLeu Pro Gly 90 95 100 gtt tct ttg ggt tat aaa ata tac gat gcc tgt aactct ata gca ttg 392 Val Ser Leu Gly Tyr Lys Ile Tyr Asp Ala Cys Asn SerIle Ala Leu 105 110 115 gct atc ctc tca ggc atg tct ttg atg aat ggt tatgaa aat att ttg 440 Ala Ile Leu Ser Gly Met Ser Leu Met Asn Gly Tyr GluAsn Ile Leu 120 125 130 agt gat ata tcc tgc tct cga tca cca gct gtc caggcc att gtt gga 488 Ser Asp Ile Ser Cys Ser Arg Ser Pro Ala Val Gln AlaIle Val Gly 135 140 145 150 gag tcg aca tct tct cct acc ata gct ttg gctact gtg gtt ggg gca 536 Glu Ser Thr Ser Ser Pro Thr Ile Ala Leu Ala ThrVal Val Gly Ala 155 160 165 ttc aac ata cct gtt atc agt cat ttt gcc acatgc acg tgc ctg aat 584 Phe Asn Ile Pro Val Ile Ser His Phe Ala Thr CysThr Cys Leu Asn 170 175 180 aac agg aaa ata tat cca tcc ttc ttt aga acaata ccc agt gat tat 632 Asn Arg Lys Ile Tyr Pro Ser Phe Phe Arg Thr IlePro Ser Asp Tyr 185 190 195 tac caa agc aga gcg ctg gca cag ctt gtc aagtat ttt ggc tgg acc 680 Tyr Gln Ser Arg Ala Leu Ala Gln Leu Val Lys TyrPhe Gly Trp Thr 200 205 210 tgg gtt ggg acg gtc agg agt cgc agt gac tatggt aat aat ggg ata 728 Trp Val Gly Thr Val Arg Ser Arg Ser Asp Tyr GlyAsn Asn Gly Ile 215 220 225 230 gca gca ttt gaa gag gct gca aaa caa gaaggt att tgc att gaa tat 776 Ala Ala Phe Glu Glu Ala Ala Lys Gln Glu GlyIle Cys Ile Glu Tyr 235 240 245 tca gaa gct gta tta aga act gat cca ccagag cag ttt ctg aag aca 824 Ser Glu Ala Val Leu Arg Thr Asp Pro Pro GluGln Phe Leu Lys Thr 250 255 260 ctg gaa gtg att aaa aag ggc aca gcc agggtt gtg gtg gct ttt atc 872 Leu Glu Val Ile Lys Lys Gly Thr Ala Arg ValVal Val Ala Phe Ile 265 270 275 tca ttt gga gat ttt gcc ccc ctt gtg aaagta att gca gaa caa aac 920 Ser Phe Gly Asp Phe Ala Pro Leu Val Lys ValIle Ala Glu Gln Asn 280 285 290 atc aca ggg ctg cag tgg gtt ggc agt gaatcc tgg ata aca tct cga 968 Ile Thr Gly Leu Gln Trp Val Gly Ser Glu SerTrp Ile Thr Ser Arg 295 300 305 310 aat ctt gca gaa acc aag gaa tac agtttc ctt tct gga gct gtg ggc 1016 Asn Leu Ala Glu Thr Lys Glu Tyr Ser PheLeu Ser Gly Ala Val Gly 315 320 325 ttt gct gta gta aat gcc aag ctt ctgggt ctg cga gag ttc cta gtg 1064 Phe Ala Val Val Asn Ala Lys Leu Leu GlyLeu Arg Glu Phe Leu Val 330 335 340 aat gtg aac cct aat caa gaa cta aaaaat gaa ctt tta aag gaa ttc 1112 Asn Val Asn Pro Asn Gln Glu Leu Lys AsnGlu Leu Leu Lys Glu Phe 345 350 355 tgg gaa aca gct ttt cag tgt tct ttcaga tcc agt ggt agt aat gcc 1160 Trp Glu Thr Ala Phe Gln Cys Ser Phe ArgSer Ser Gly Ser Asn Ala 360 365 370 tgt act ggc tca gag aaa ctg gca gagctg caa aat gaa tat act gat 1208 Cys Thr Gly Ser Glu Lys Leu Ala Glu LeuGln Asn Glu Tyr Thr Asp 375 380 385 390 gta tct gag cta cga ata gaa cataaa gct tac act gca gtg tat gct 1256 Val Ser Glu Leu Arg Ile Glu His LysAla Tyr Thr Ala Val Tyr Ala 395 400 405 gtt gca cac aca ctg cat aat gtttta aaa gac ttt aaa tca tcc acc 1304 Val Ala His Thr Leu His Asn Val LeuLys Asp Phe Lys Ser Ser Thr 410 415 420 aac aac agc aaa gga gag ctg cccaca cca aaa aaa gta ttg caa tat 1352 Asn Asn Ser Lys Gly Glu Leu Pro ThrPro Lys Lys Val Leu Gln Tyr 425 430 435 atg aga gat gtg agc ttc act atgaaa aca ggt gag aat ata ttt ttt 1400 Met Arg Asp Val Ser Phe Thr Met LysThr Gly Glu Asn Ile Phe Phe 440 445 450 gat gca agt ggt gat cca gtg gcaata tat gac ctg gtg aac tgg cag 1448 Asp Ala Ser Gly Asp Pro Val Ala IleTyr Asp Leu Val Asn Trp Gln 455 460 465 470 cct gct gag gat gga aga ttacag ttc gag aat gtg ggt gtc tat gac 1496 Pro Ala Glu Asp Gly Arg Leu GlnPhe Glu Asn Val Gly Val Tyr Asp 475 480 485 agc tca ctg cct tta gag caacgt ctt caa gtt aat cag gaa cac ata 1544 Ser Ser Leu Pro Leu Glu Gln ArgLeu Gln Val Asn Gln Glu His Ile 490 495 500 cta tgg gca ggg aag aga gcacag ttg cct ggg tcc gtg tgc agt gaa 1592 Leu Trp Ala Gly Lys Arg Ala GlnLeu Pro Gly Ser Val Cys Ser Glu 505 510 515 agc tgc ccc act gga act agaaag act gtg cag aaa ggt cgg cct gtt 1640 Ser Cys Pro Thr Gly Thr Arg LysThr Val Gln Lys Gly Arg Pro Val 520 525 530 tgc tgt tat gac tgt act ccatgt gca gaa gga gaa atc agt aat agc 1688 Cys Cys Tyr Asp Cys Thr Pro CysAla Glu Gly Glu Ile Ser Asn Ser 535 540 545 550 aca gat tct agt gac tgcttt cct tgt gat ttg gag tac tgg tcg aat 1736 Thr Asp Ser Ser Asp Cys PhePro Cys Asp Leu Glu Tyr Trp Ser Asn 555 560 565 gaa agc aga gac aga tgtgta tta aaa gtg gtt gaa ttc ctt tcc tat 1784 Glu Ser Arg Asp Arg Cys ValLeu Lys Val Val Glu Phe Leu Ser Tyr 570 575 580 aca gaa atc atg ggg atggtg ctt tgc att ttc tcc ttc att ggc gta 1832 Thr Glu Ile Met Gly Met ValLeu Cys Ile Phe Ser Phe Ile Gly Val 585 590 595 tta tta aca gca atg gtatct ttt ctg ttt tat ctc cat aaa gaa aca 1880 Leu Leu Thr Ala Met Val SerPhe Leu Phe Tyr Leu His Lys Glu Thr 600 605 610 cct att gta aga gcc aacaac tca gag ctg agc ttc ctg ttg ctc ttc 1928 Pro Ile Val Arg Ala Asn AsnSer Glu Leu Ser Phe Leu Leu Leu Phe 615 620 625 630 tca ctc tca ctg tgtttt ctc tgt tca cta act ttc att ggc cgg ccc 1976 Ser Leu Ser Leu Cys PheLeu Cys Ser Leu Thr Phe Ile Gly Arg Pro 635 640 645 act gag ctg tcc tgtatg ttg cgt cac aca gca ttt ggg atc act ttt 2024 Thr Glu Leu Ser Cys MetLeu Arg His Thr Ala Phe Gly Ile Thr Phe 650 655 660 gtc ctt tgt atc tcctgt gtt ctg ggg aaa aca ttg gta gtt tta atg 2072 Val Leu Cys Ile Ser CysVal Leu Gly Lys Thr Leu Val Val Leu Met 665 670 675 gcc ttc aga gct acgctt cca gga agt gat gtc atg aaa tgg ttt ggg 2120 Ala Phe Arg Ala Thr LeuPro Gly Ser Asp Val Met Lys Trp Phe Gly 680 685 690 cct gca cag cag cgactc agt gtt gtt tcc tta aca tta ata cag gtg 2168 Pro Ala Gln Gln Arg LeuSer Val Val Ser Leu Thr Leu Ile Gln Val 695 700 705 710 att gtc tgt gtgctt tgg tta aca ata tcc cct cct ttc cca tat atg 2216 Ile Val Cys Val LeuTrp Leu Thr Ile Ser Pro Pro Phe Pro Tyr Met 715 720 725 aat tta agc tattat aga gaa aaa ata att cta gaa tgt aat gta ggt 2264 Asn Leu Ser Tyr TyrArg Glu Lys Ile Ile Leu Glu Cys Asn Val Gly 730 735 740 tca gct ctt ggtttc tgg act gtt ctg tgt tat act ggc ctg cta tca 2312 Ser Ala Leu Gly PheTrp Thr Val Leu Cys Tyr Thr Gly Leu Leu Ser 745 750 755 agc ttg tgt tttgtt tta gct ttt ctt gct cgg aag ctc cct gat aac 2360 Ser Leu Cys Phe ValLeu Ala Phe Leu Ala Arg Lys Leu Pro Asp Asn 760 765 770 ttc aat gag gccaag ttc atc aca ttc agc atg ctc ata ttc tgt gct 2408 Phe Asn Glu Ala LysPhe Ile Thr Phe Ser Met Leu Ile Phe Cys Ala 775 780 785 790 gtc tgg ctcaca ttt atc cca gct tat gtc agt tct cct gga aaa ttt 2456 Val Trp Leu ThrPhe Ile Pro Ala Tyr Val Ser Ser Pro Gly Lys Phe 795 800 805 act gta gctgtg gag ata ttt gcc att tta gtt tca agt ttt ggt tta 2504 Thr Val Ala ValGlu Ile Phe Ala Ile Leu Val Ser Ser Phe Gly Leu 810 815 820 cta ttt tgcata ttt gcc cca aaa tgt tac ata att ttg cta aaa cca 2552 Leu Phe Cys IlePhe Ala Pro Lys Cys Tyr Ile Ile Leu Leu Lys Pro 825 830 835 gag aaa aacaca aag aaa caa atg atg ggg aaa tct tct aca gct ctt 2600 Glu Lys Asn ThrLys Lys Gln Met Met Gly Lys Ser Ser Thr Ala Leu 840 845 850 tgaaacaagagttaatcata taactgatta aaagccaaca tagccagctc tatcaaatgc 2660 atttctcccacaggctgtgg caggctctgc agtgtaggcc gggtcagcac tagacacagt 2720 ggactctcagtgaccatctc catcacagcc atattctgga cctgcattat gtactttcta 2780 ataagaaatgatattgactt ttygcagtta acaacaaatg gaagttactt tttaatgtat 2840 tgaatttatatctttagttt tctagatttt ctacatmact gttgttttaa cmgtaaatag 2900 tatagacmttgtggsggcsc ccc 2923 10 854 PRT Carassius auratus 10 Met Ala Lys Ser ThrVal Ser Leu Leu Leu Leu Leu Val Val Val His 1 5 10 15 Gly Val Phe ValPro Ala Ser Ala Gln Leu Cys Ser Leu Leu Gly His 20 25 30 Ser Ala Phe ProVal Leu Ser Ala Glu Arg Asp Ile Asn Ile Gly Ala 35 40 45 Ile Phe Ser IleHis Arg Ser Ala Leu Leu Lys Met His Pro Phe Thr 50 55 60 Ser Lys Pro GluPro Thr Thr Cys Leu Arg Leu Asn Leu Arg Glu Phe 65 70 75 80 Lys Phe AlaGln Thr Phe Ile Phe Ala Ile Glu Glu Ile Asn Asn Ser 85 90 95 Thr Gln LeuLeu Pro Gly Val Ser Leu Gly Tyr Lys Ile Tyr Asp Ala 100 105 110 Cys AsnSer Ile Ala Leu Ala Ile Leu Ser Gly Met Ser Leu Met Asn 115 120 125 GlyTyr Glu Asn Ile Leu Ser Asp Ile Ser Cys Ser Arg Ser Pro Ala 130 135 140Val Gln Ala Ile Val Gly Glu Ser Thr Ser Ser Pro Thr Ile Ala Leu 145 150155 160 Ala Thr Val Val Gly Ala Phe Asn Ile Pro Val Ile Ser His Phe Ala165 170 175 Thr Cys Thr Cys Leu Asn Asn Arg Lys Ile Tyr Pro Ser Phe PheArg 180 185 190 Thr Ile Pro Ser Asp Tyr Tyr Gln Ser Arg Ala Leu Ala GlnLeu Val 195 200 205 Lys Tyr Phe Gly Trp Thr Trp Val Gly Thr Val Arg SerArg Ser Asp 210 215 220 Tyr Gly Asn Asn Gly Ile Ala Ala Phe Glu Glu AlaAla Lys Gln Glu 225 230 235 240 Gly Ile Cys Ile Glu Tyr Ser Glu Ala ValLeu Arg Thr Asp Pro Pro 245 250 255 Glu Gln Phe Leu Lys Thr Leu Glu ValIle Lys Lys Gly Thr Ala Arg 260 265 270 Val Val Val Ala Phe Ile Ser PheGly Asp Phe Ala Pro Leu Val Lys 275 280 285 Val Ile Ala Glu Gln Asn IleThr Gly Leu Gln Trp Val Gly Ser Glu 290 295 300 Ser Trp Ile Thr Ser ArgAsn Leu Ala Glu Thr Lys Glu Tyr Ser Phe 305 310 315 320 Leu Ser Gly AlaVal Gly Phe Ala Val Val Asn Ala Lys Leu Leu Gly 325 330 335 Leu Arg GluPhe Leu Val Asn Val Asn Pro Asn Gln Glu Leu Lys Asn 340 345 350 Glu LeuLeu Lys Glu Phe Trp Glu Thr Ala Phe Gln Cys Ser Phe Arg 355 360 365 SerSer Gly Ser Asn Ala Cys Thr Gly Ser Glu Lys Leu Ala Glu Leu 370 375 380Gln Asn Glu Tyr Thr Asp Val Ser Glu Leu Arg Ile Glu His Lys Ala 385 390395 400 Tyr Thr Ala Val Tyr Ala Val Ala His Thr Leu His Asn Val Leu Lys405 410 415 Asp Phe Lys Ser Ser Thr Asn Asn Ser Lys Gly Glu Leu Pro ThrPro 420 425 430 Lys Lys Val Leu Gln Tyr Met Arg Asp Val Ser Phe Thr MetLys Thr 435 440 445 Gly Glu Asn Ile Phe Phe Asp Ala Ser Gly Asp Pro ValAla Ile Tyr 450 455 460 Asp Leu Val Asn Trp Gln Pro Ala Glu Asp Gly ArgLeu Gln Phe Glu 465 470 475 480 Asn Val Gly Val Tyr Asp Ser Ser Leu ProLeu Glu Gln Arg Leu Gln 485 490 495 Val Asn Gln Glu His Ile Leu Trp AlaGly Lys Arg Ala Gln Leu Pro 500 505 510 Gly Ser Val Cys Ser Glu Ser CysPro Thr Gly Thr Arg Lys Thr Val 515 520 525 Gln Lys Gly Arg Pro Val CysCys Tyr Asp Cys Thr Pro Cys Ala Glu 530 535 540 Gly Glu Ile Ser Asn SerThr Asp Ser Ser Asp Cys Phe Pro Cys Asp 545 550 555 560 Leu Glu Tyr TrpSer Asn Glu Ser Arg Asp Arg Cys Val Leu Lys Val 565 570 575 Val Glu PheLeu Ser Tyr Thr Glu Ile Met Gly Met Val Leu Cys Ile 580 585 590 Phe SerPhe Ile Gly Val Leu Leu Thr Ala Met Val Ser Phe Leu Phe 595 600 605 TyrLeu His Lys Glu Thr Pro Ile Val Arg Ala Asn Asn Ser Glu Leu 610 615 620Ser Phe Leu Leu Leu Phe Ser Leu Ser Leu Cys Phe Leu Cys Ser Leu 625 630635 640 Thr Phe Ile Gly Arg Pro Thr Glu Leu Ser Cys Met Leu Arg His Thr645 650 655 Ala Phe Gly Ile Thr Phe Val Leu Cys Ile Ser Cys Val Leu GlyLys 660 665 670 Thr Leu Val Val Leu Met Ala Phe Arg Ala Thr Leu Pro GlySer Asp 675 680 685 Val Met Lys Trp Phe Gly Pro Ala Gln Gln Arg Leu SerVal Val Ser 690 695 700 Leu Thr Leu Ile Gln Val Ile Val Cys Val Leu TrpLeu Thr Ile Ser 705 710 715 720 Pro Pro Phe Pro Tyr Met Asn Leu Ser TyrTyr Arg Glu Lys Ile Ile 725 730 735 Leu Glu Cys Asn Val Gly Ser Ala LeuGly Phe Trp Thr Val Leu Cys 740 745 750 Tyr Thr Gly Leu Leu Ser Ser LeuCys Phe Val Leu Ala Phe Leu Ala 755 760 765 Arg Lys Leu Pro Asp Asn PheAsn Glu Ala Lys Phe Ile Thr Phe Ser 770 775 780 Met Leu Ile Phe Cys AlaVal Trp Leu Thr Phe Ile Pro Ala Tyr Val 785 790 795 800 Ser Ser Pro GlyLys Phe Thr Val Ala Val Glu Ile Phe Ala Ile Leu 805 810 815 Val Ser SerPhe Gly Leu Leu Phe Cys Ile Phe Ala Pro Lys Cys Tyr 820 825 830 Ile IleLeu Leu Lys Pro Glu Lys Asn Thr Lys Lys Gln Met Met Gly 835 840 845 LysSer Ser Thr Ala Leu 850 11 2749 DNA Carassius auratus CDS (19)..(2571)11 cgacccacgc gtccggac atg gca aag tgg act tta tca gtg ctg caa ctg 51Met Ala Lys Trp Thr Leu Ser Val Leu Gln Leu 1 5 10 ctg ctg gtg gtg tatggg gtc agt gtg cct gca tta gcg caa atc tgc 99 Leu Leu Val Val Tyr GlyVal Ser Val Pro Ala Leu Ala Gln Ile Cys 15 20 25 aga ctg ctt ggt cag cctgcc ctt cct cta ctt tct gca caa aaa gac 147 Arg Leu Leu Gly Gln Pro AlaLeu Pro Leu Leu Ser Ala Gln Lys Asp 30 35 40 att aat att ggg gca att ttctca ttt cac aaa agt gct ctg ctg aag 195 Ile Asn Ile Gly Ala Ile Phe SerPhe His Lys Ser Ala Leu Leu Lys 45 50 55 atc cag cct ttc act tct aaa ccaaat cca aca aca tgc ggc agc ttc 243 Ile Gln Pro Phe Thr Ser Lys Pro AsnPro Thr Thr Cys Gly Ser Phe 60 65 70 75 aac agc tta cgt ggg ttt aag tatgct cag aca ctc ata ttt aca att 291 Asn Ser Leu Arg Gly Phe Lys Tyr AlaGln Thr Leu Ile Phe Thr Ile 80 85 90 gag gag att aat aac agc aaa cag ctgttg cct ggt gtt tct ttg ggc 339 Glu Glu Ile Asn Asn Ser Lys Gln Leu LeuPro Gly Val Ser Leu Gly 95 100 105 tac aag ata tat gat tcc tgt agc tctata tct caa act gtt ctg tca 387 Tyr Lys Ile Tyr Asp Ser Cys Ser Ser IleSer Gln Thr Val Leu Ser 110 115 120 ggc atg tct tta atg aat gga tat gaagag act ttg aat gat aca tcc 435 Gly Met Ser Leu Met Asn Gly Tyr Glu GluThr Leu Asn Asp Thr Ser 125 130 135 tgc tct aga cca cca gct gtt cat gccatt gtt gga gaa tca aac tcc 483 Cys Ser Arg Pro Pro Ala Val His Ala IleVal Gly Glu Ser Asn Ser 140 145 150 155 tct ccc acc atg gca ctg gct tctata gtt ggt cct ttc agc tta ccc 531 Ser Pro Thr Met Ala Leu Ala Ser IleVal Gly Pro Phe Ser Leu Pro 160 165 170 gtt att agt cat ttt gcc aca tgtgca tgc ctg agt aac aga aaa agg 579 Val Ile Ser His Phe Ala Thr Cys AlaCys Leu Ser Asn Arg Lys Arg 175 180 185 ttt ccg tca ttc ttc aga aca ataccc agt gat tat tat caa agc aga 627 Phe Pro Ser Phe Phe Arg Thr Ile ProSer Asp Tyr Tyr Gln Ser Arg 190 195 200 gct ctc gct cag ctt gtc aag cacttt ggc tgg acc tgg gtt ggg aca 675 Ala Leu Ala Gln Leu Val Lys His PheGly Trp Thr Trp Val Gly Thr 205 210 215 gtc agg agt cgt gga gac tat ggcaat aat ggt att tca gca ttt gag 723 Val Arg Ser Arg Gly Asp Tyr Gly AsnAsn Gly Ile Ser Ala Phe Glu 220 225 230 235 gag gct gca aga caa gaa gggatt tgt att gaa tac tca gag gcc ata 771 Glu Ala Ala Arg Gln Glu Gly IleCys Ile Glu Tyr Ser Glu Ala Ile 240 245 250 tta agc aca gat cca aag gagcag ttt tta aag aca cta gaa gtg ata 819 Leu Ser Thr Asp Pro Lys Glu GlnPhe Leu Lys Thr Leu Glu Val Ile 255 260 265 aag aag ggc act gcc aag gtagtg ctg gct ttc gtt gca gta gga gat 867 Lys Lys Gly Thr Ala Lys Val ValLeu Ala Phe Val Ala Val Gly Asp 270 275 280 ttt gtt ccc ctc tta aat gtaatt gcg caa cac aac atc aca ggg att 915 Phe Val Pro Leu Leu Asn Val IleAla Gln His Asn Ile Thr Gly Ile 285 290 295 cag tgg gtt ggc agt gaa tcttgg atc act tat cga aca ttt gca gaa 963 Gln Trp Val Gly Ser Glu Ser TrpIle Thr Tyr Arg Thr Phe Ala Glu 300 305 310 315 aca aaa gaa tac agt ttcctc tct gga gct gtg ggt ttt gct ata gca 1011 Thr Lys Glu Tyr Ser Phe LeuSer Gly Ala Val Gly Phe Ala Ile Ala 320 325 330 aat gct aaa ctt gtg ggcctg aga gag ttc cta gta aat gtg cat cct 1059 Asn Ala Lys Leu Val Gly LeuArg Glu Phe Leu Val Asn Val His Pro 335 340 345 gat caa gaa cca aac aataaa ctt tta aaa gaa ttc tgg gaa aca gtt 1107 Asp Gln Glu Pro Asn Asn LysLeu Leu Lys Glu Phe Trp Glu Thr Val 350 355 360 ttt cag tgc tct ttc agaagc aac agt agt ggt ggc tgt act ggc tcc 1155 Phe Gln Cys Ser Phe Arg SerAsn Ser Ser Gly Gly Cys Thr Gly Ser 365 370 375 gaa aaa ctg gca gag ctgcaa aat gaa tat act gat gta tca gag cta 1203 Glu Lys Leu Ala Glu Leu GlnAsn Glu Tyr Thr Asp Val Ser Glu Leu 380 385 390 395 cgg att gta aat aaagtg tac act gca gtg tat gct att gca cat aca 1251 Arg Ile Val Asn Lys ValTyr Thr Ala Val Tyr Ala Ile Ala His Thr 400 405 410 cta cac aat gta ttaaaa gac ttg aga tcc tcc acc aac agc agc aaa 1299 Leu His Asn Val Leu LysAsp Leu Arg Ser Ser Thr Asn Ser Ser Lys 415 420 425 gga gaa tgg cct acacta caa aag gtg ttg aat tat atg agg gat gtg 1347 Gly Glu Trp Pro Thr LeuGln Lys Val Leu Asn Tyr Met Arg Asp Val 430 435 440 aga ttc act gtt aaaaca ggt gaa gaa atc ttc ttt gat tta agt ggt 1395 Arg Phe Thr Val Lys ThrGly Glu Glu Ile Phe Phe Asp Leu Ser Gly 445 450 455 gat cca gca gcg agatat gac ctt att aac tgg cag cct gct gaa aat 1443 Asp Pro Ala Ala Arg TyrAsp Leu Ile Asn Trp Gln Pro Ala Glu Asn 460 465 470 475 gga agt ttg cagttt aag tat gtg ggc tca tat gac agc tca ctg cca 1491 Gly Ser Leu Gln PheLys Tyr Val Gly Ser Tyr Asp Ser Ser Leu Pro 480 485 490 ttc gaa cag tgtctt caa gtc acc cag gaa caa atg ata tgg gca ggg 1539 Phe Glu Gln Cys LeuGln Val Thr Gln Glu Gln Met Ile Trp Ala Gly 495 500 505 aac agt agg cagttc cct gtg tcc gtg tgc agt gag agc tgc ccc cca 1587 Asn Ser Arg Gln PhePro Val Ser Val Cys Ser Glu Ser Cys Pro Pro 510 515 520 ggt act aga aaagct gtg caa aag ggg cga cct gtt tgc tgc tat gac 1635 Gly Thr Arg Lys AlaVal Gln Lys Gly Arg Pro Val Cys Cys Tyr Asp 525 530 535 tgt att cca tgttcg gaa gga gaa ata aat aat gaa aca gat tct agt 1683 Cys Ile Pro Cys SerGlu Gly Glu Ile Asn Asn Glu Thr Asp Ser Ser 540 545 550 555 gac tgc tttcct tgt gat ttg gag tac tgg tcg aat gaa ggc aaa gac 1731 Asp Cys Phe ProCys Asp Leu Glu Tyr Trp Ser Asn Glu Gly Lys Asp 560 565 570 aaa tgt gtatta aaa gtg gta gag ttc cta tcc tat aca gaa atc atg 1779 Lys Cys Val LeuLys Val Val Glu Phe Leu Ser Tyr Thr Glu Ile Met 575 580 585 ggg acg gtgctt tgt att ttc tcc ttc ttt ggg atg tta tta aca gca 1827 Gly Thr Val LeuCys Ile Phe Ser Phe Phe Gly Met Leu Leu Thr Ala 590 595 600 att gta tctttt gtg ttt tat ctt cat aaa gaa acc cct att gtc aga 1875 Ile Val Ser PheVal Phe Tyr Leu His Lys Glu Thr Pro Ile Val Arg 605 610 615 gcc aac aactca gag ctg agc ttc ctg ctg ctc ttc tca ctc tca ctg 1923 Ala Asn Asn SerGlu Leu Ser Phe Leu Leu Leu Phe Ser Leu Ser Leu 620 625 630 635 tgt ttcctc tgt tca ctt act ttc att ggt agg cct act gag tgg tcc 1971 Cys Phe LeuCys Ser Leu Thr Phe Ile Gly Arg Pro Thr Glu Trp Ser 640 645 650 tgt atgttg cgc cac aca gca ttt ggg gtc act ttt gtc ctc tgt atc 2019 Cys Met LeuArg His Thr Ala Phe Gly Val Thr Phe Val Leu Cys Ile 655 660 665 tcc tgtgtt ttg gga aaa aca ata gtg gtc tta atg gct ttc agg gct 2067 Ser Cys ValLeu Gly Lys Thr Ile Val Val Leu Met Ala Phe Arg Ala 670 675 680 aca cttcca gga agt aat gtt atg aaa tgt ttt ggg cct ctt caa cag 2115 Thr Leu ProGly Ser Asn Val Met Lys Cys Phe Gly Pro Leu Gln Gln 685 690 695 cga ttcagt gtt gtt tca tta tca tta ata cag atg ata ata tgt gtg 2163 Arg Phe SerVal Val Ser Leu Ser Leu Ile Gln Met Ile Ile Cys Val 700 705 710 715 ctttgg tta aca ata tcc cca cct ttt cct ttt atg aat ttg agc tat 2211 Leu TrpLeu Thr Ile Ser Pro Pro Phe Pro Phe Met Asn Leu Ser Tyr 720 725 730 tacaga gaa aag atc atc cta gaa tgt aac tta ggt tca gct ctt ggc 2259 Tyr ArgGlu Lys Ile Ile Leu Glu Cys Asn Leu Gly Ser Ala Leu Gly 735 740 745 ttctgg ggt gtt ctg ggt tat act ggc ttg cta tca att ttg tgt ttt 2307 Phe TrpGly Val Leu Gly Tyr Thr Gly Leu Leu Ser Ile Leu Cys Phe 750 755 760 atttta gct ttt ctt gct agg aaa ctc cct gat aat ttc aac gag gcc 2355 Ile LeuAla Phe Leu Ala Arg Lys Leu Pro Asp Asn Phe Asn Glu Ala 765 770 775 aagttc ata aca ttc agt atg ctc ata ttc tgt gct gta tgg atc aca 2403 Lys PheIle Thr Phe Ser Met Leu Ile Phe Cys Ala Val Trp Ile Thr 780 785 790 795ttt att cca gct tat gtc agt tct cct gga aaa ttt act gta gcc gtg 2451 PheIle Pro Ala Tyr Val Ser Ser Pro Gly Lys Phe Thr Val Ala Val 800 805 810cag ata ttt gct att tta gca tca agt ttt agt tta ctc ttt tgc ata 2499 GlnIle Phe Ala Ile Leu Ala Ser Ser Phe Ser Leu Leu Phe Cys Ile 815 820 825ttt gct cca aaa tgt tac att att ttg cta aaa cca gag aaa aat aca 2547 PheAla Pro Lys Cys Tyr Ile Ile Leu Leu Lys Pro Glu Lys Asn Thr 830 835 840aag aaa caa ata atg ggg aaa tct taatctaaag ctctttaagc tcagagataa 2601Lys Lys Gln Ile Met Gly Lys Ser 845 850 ttgtgtaaat cacaaaaatg taaagaaaacttaatatatt cctctgtatt ctgagaatta 2661 aactagcaga taagtgaata caagtattttgcctataaaa aaagtaaaaa gaaaacctaa 2721 agaaaaaaaa aaaaaaaaaa agggcggc2749 12 851 PRT Carassius auratus 12 Met Ala Lys Trp Thr Leu Ser Val LeuGln Leu Leu Leu Val Val Tyr 1 5 10 15 Gly Val Ser Val Pro Ala Leu AlaGln Ile Cys Arg Leu Leu Gly Gln 20 25 30 Pro Ala Leu Pro Leu Leu Ser AlaGln Lys Asp Ile Asn Ile Gly Ala 35 40 45 Ile Phe Ser Phe His Lys Ser AlaLeu Leu Lys Ile Gln Pro Phe Thr 50 55 60 Ser Lys Pro Asn Pro Thr Thr CysGly Ser Phe Asn Ser Leu Arg Gly 65 70 75 80 Phe Lys Tyr Ala Gln Thr LeuIle Phe Thr Ile Glu Glu Ile Asn Asn 85 90 95 Ser Lys Gln Leu Leu Pro GlyVal Ser Leu Gly Tyr Lys Ile Tyr Asp 100 105 110 Ser Cys Ser Ser Ile SerGln Thr Val Leu Ser Gly Met Ser Leu Met 115 120 125 Asn Gly Tyr Glu GluThr Leu Asn Asp Thr Ser Cys Ser Arg Pro Pro 130 135 140 Ala Val His AlaIle Val Gly Glu Ser Asn Ser Ser Pro Thr Met Ala 145 150 155 160 Leu AlaSer Ile Val Gly Pro Phe Ser Leu Pro Val Ile Ser His Phe 165 170 175 AlaThr Cys Ala Cys Leu Ser Asn Arg Lys Arg Phe Pro Ser Phe Phe 180 185 190Arg Thr Ile Pro Ser Asp Tyr Tyr Gln Ser Arg Ala Leu Ala Gln Leu 195 200205 Val Lys His Phe Gly Trp Thr Trp Val Gly Thr Val Arg Ser Arg Gly 210215 220 Asp Tyr Gly Asn Asn Gly Ile Ser Ala Phe Glu Glu Ala Ala Arg Gln225 230 235 240 Glu Gly Ile Cys Ile Glu Tyr Ser Glu Ala Ile Leu Ser ThrAsp Pro 245 250 255 Lys Glu Gln Phe Leu Lys Thr Leu Glu Val Ile Lys LysGly Thr Ala 260 265 270 Lys Val Val Leu Ala Phe Val Ala Val Gly Asp PheVal Pro Leu Leu 275 280 285 Asn Val Ile Ala Gln His Asn Ile Thr Gly IleGln Trp Val Gly Ser 290 295 300 Glu Ser Trp Ile Thr Tyr Arg Thr Phe AlaGlu Thr Lys Glu Tyr Ser 305 310 315 320 Phe Leu Ser Gly Ala Val Gly PheAla Ile Ala Asn Ala Lys Leu Val 325 330 335 Gly Leu Arg Glu Phe Leu ValAsn Val His Pro Asp Gln Glu Pro Asn 340 345 350 Asn Lys Leu Leu Lys GluPhe Trp Glu Thr Val Phe Gln Cys Ser Phe 355 360 365 Arg Ser Asn Ser SerGly Gly Cys Thr Gly Ser Glu Lys Leu Ala Glu 370 375 380 Leu Gln Asn GluTyr Thr Asp Val Ser Glu Leu Arg Ile Val Asn Lys 385 390 395 400 Val TyrThr Ala Val Tyr Ala Ile Ala His Thr Leu His Asn Val Leu 405 410 415 LysAsp Leu Arg Ser Ser Thr Asn Ser Ser Lys Gly Glu Trp Pro Thr 420 425 430Leu Gln Lys Val Leu Asn Tyr Met Arg Asp Val Arg Phe Thr Val Lys 435 440445 Thr Gly Glu Glu Ile Phe Phe Asp Leu Ser Gly Asp Pro Ala Ala Arg 450455 460 Tyr Asp Leu Ile Asn Trp Gln Pro Ala Glu Asn Gly Ser Leu Gln Phe465 470 475 480 Lys Tyr Val Gly Ser Tyr Asp Ser Ser Leu Pro Phe Glu GlnCys Leu 485 490 495 Gln Val Thr Gln Glu Gln Met Ile Trp Ala Gly Asn SerArg Gln Phe 500 505 510 Pro Val Ser Val Cys Ser Glu Ser Cys Pro Pro GlyThr Arg Lys Ala 515 520 525 Val Gln Lys Gly Arg Pro Val Cys Cys Tyr AspCys Ile Pro Cys Ser 530 535 540 Glu Gly Glu Ile Asn Asn Glu Thr Asp SerSer Asp Cys Phe Pro Cys 545 550 555 560 Asp Leu Glu Tyr Trp Ser Asn GluGly Lys Asp Lys Cys Val Leu Lys 565 570 575 Val Val Glu Phe Leu Ser TyrThr Glu Ile Met Gly Thr Val Leu Cys 580 585 590 Ile Phe Ser Phe Phe GlyMet Leu Leu Thr Ala Ile Val Ser Phe Val 595 600 605 Phe Tyr Leu His LysGlu Thr Pro Ile Val Arg Ala Asn Asn Ser Glu 610 615 620 Leu Ser Phe LeuLeu Leu Phe Ser Leu Ser Leu Cys Phe Leu Cys Ser 625 630 635 640 Leu ThrPhe Ile Gly Arg Pro Thr Glu Trp Ser Cys Met Leu Arg His 645 650 655 ThrAla Phe Gly Val Thr Phe Val Leu Cys Ile Ser Cys Val Leu Gly 660 665 670Lys Thr Ile Val Val Leu Met Ala Phe Arg Ala Thr Leu Pro Gly Ser 675 680685 Asn Val Met Lys Cys Phe Gly Pro Leu Gln Gln Arg Phe Ser Val Val 690695 700 Ser Leu Ser Leu Ile Gln Met Ile Ile Cys Val Leu Trp Leu Thr Ile705 710 715 720 Ser Pro Pro Phe Pro Phe Met Asn Leu Ser Tyr Tyr Arg GluLys Ile 725 730 735 Ile Leu Glu Cys Asn Leu Gly Ser Ala Leu Gly Phe TrpGly Val Leu 740 745 750 Gly Tyr Thr Gly Leu Leu Ser Ile Leu Cys Phe IleLeu Ala Phe Leu 755 760 765 Ala Arg Lys Leu Pro Asp Asn Phe Asn Glu AlaLys Phe Ile Thr Phe 770 775 780 Ser Met Leu Ile Phe Cys Ala Val Trp IleThr Phe Ile Pro Ala Tyr 785 790 795 800 Val Ser Ser Pro Gly Lys Phe ThrVal Ala Val Gln Ile Phe Ala Ile 805 810 815 Leu Ala Ser Ser Phe Ser LeuLeu Phe Cys Ile Phe Ala Pro Lys Cys 820 825 830 Tyr Ile Ile Leu Leu LysPro Glu Lys Asn Thr Lys Lys Gln Ile Met 835 840 845 Gly Lys Ser 850 132595 DNA Brachydanio rerio (zebrafish) CDS (4)..(2592) 13 aac atg gatttg atg agc ttc att ctc tta tgg gct ggg ctg atg aaa 48 Met Asp Leu MetSer Phe Ile Leu Leu Trp Ala Gly Leu Met Lys 1 5 10 15 gtc gca gaa gcctca att gca cag ttc agc cag ttg gga gcc tca gcc 96 Val Ala Glu Ala SerIle Ala Gln Phe Ser Gln Leu Gly Ala Ser Ala 20 25 30 cct gga aac atc atcatt gga gga ctt ttc ccc atc cat gag gca gtg 144 Pro Gly Asn Ile Ile IleGly Gly Leu Phe Pro Ile His Glu Ala Val 35 40 45 gtg cca gta aac tac accggc aac aac agc atc tct gcc cct gag cat 192 Val Pro Val Asn Tyr Thr GlyAsn Asn Ser Ile Ser Ala Pro Glu His 50 55 60 ccg gac tgc atc aga ttc tacaca aag ggt cta aat caa gct cta gcg 240 Pro Asp Cys Ile Arg Phe Tyr ThrLys Gly Leu Asn Gln Ala Leu Ala 65 70 75 atg att aat gct gta gaa atg gcaaac aaa tcc ccc atg ttg agc agt 288 Met Ile Asn Ala Val Glu Met Ala AsnLys Ser Pro Met Leu Ser Ser 80 85 90 95 ttg aac att act cta gga tac cgaatc tac gac aca tgt tct gat gtc 336 Leu Asn Ile Thr Leu Gly Tyr Arg IleTyr Asp Thr Cys Ser Asp Val 100 105 110 acg act gca ctg cgg gct gtc catgat att atg agg ccg ttc tca gac 384 Thr Thr Ala Leu Arg Ala Val His AspIle Met Arg Pro Phe Ser Asp 115 120 125 tgt gaa tca cca gaa gac tca tctcaa ccc gtc cag cca ata atg gca 432 Cys Glu Ser Pro Glu Asp Ser Ser GlnPro Val Gln Pro Ile Met Ala 130 135 140 gta att ggg acc act tca tcc gagatc tca atc gca gtt gct cga gat 480 Val Ile Gly Thr Thr Ser Ser Glu IleSer Ile Ala Val Ala Arg Asp 145 150 155 ctc aac ctt cag atg ata cct cagatt agt tac gca tct aca gcc acg 528 Leu Asn Leu Gln Met Ile Pro Gln IleSer Tyr Ala Ser Thr Ala Thr 160 165 170 175 att ttg agt gat aaa agt cgtttc cct gct ttc atg agg act gtg ccc 576 Ile Leu Ser Asp Lys Ser Arg PhePro Ala Phe Met Arg Thr Val Pro 180 185 190 agt gat gag tat caa acc tgtgcc atg gcc aaa ctt cta aag tcc aac 624 Ser Asp Glu Tyr Gln Thr Cys AlaMet Ala Lys Leu Leu Lys Ser Asn 195 200 205 aaa tgg agc tgg gtt ggc attatc att aca gat gga gat tat gga cgt 672 Lys Trp Ser Trp Val Gly Ile IleIle Thr Asp Gly Asp Tyr Gly Arg 210 215 220 tct gcc ttg gaa ggt ttc atacag cac acc gaa acg gag gga att tgc 720 Ser Ala Leu Glu Gly Phe Ile GlnHis Thr Glu Thr Glu Gly Ile Cys 225 230 235 atc gcc ttt aaa gca atc cttcca gac tca cta gca gat caa cag aaa 768 Ile Ala Phe Lys Ala Ile Leu ProAsp Ser Leu Ala Asp Gln Gln Lys 240 245 250 255 cta aac aca gac atc gaaaac acc ttg aac atc att gaa aac aat ccg 816 Leu Asn Thr Asp Ile Glu AsnThr Leu Asn Ile Ile Glu Asn Asn Pro 260 265 270 aaa gtt aga gtg gtg atctcg ttt gct aaa tcc tct caa atg cag ttg 864 Lys Val Arg Val Val Ile SerPhe Ala Lys Ser Ser Gln Met Gln Leu 275 280 285 cta ttt aag ggg ctg cagagt aga aac att tca aat aac atg gtg tgg 912 Leu Phe Lys Gly Leu Gln SerArg Asn Ile Ser Asn Asn Met Val Trp 290 295 300 gtt gcc agt gat aac tggtcg acg gct aaa cat att ctg aat gat ggt 960 Val Ala Ser Asp Asn Trp SerThr Ala Lys His Ile Leu Asn Asp Gly 305 310 315 agc atc act gat att gggaaa gtg ctg ggc ttt acc ttc aag agt gga 1008 Ser Ile Thr Asp Ile Gly LysVal Leu Gly Phe Thr Phe Lys Ser Gly 320 325 330 335 aat ttt aca tct tttcat cag tac cta aag aat cta cag ttt gaa agt 1056 Asn Phe Thr Ser Phe HisGln Tyr Leu Lys Asn Leu Gln Phe Glu Ser 340 345 350 gaa gat gag atg aacaat tca ttc ctg aag gaa ttt tta aaa ctc aac 1104 Glu Asp Glu Met Asn AsnSer Phe Leu Lys Glu Phe Leu Lys Leu Asn 355 360 365 gca ggc aat gct tccaat acc gtg ctg gag ctg atg aaa agc acc aat 1152 Ala Gly Asn Ala Ser AsnThr Val Leu Glu Leu Met Lys Ser Thr Asn 370 375 380 ttg gac aag att ttcagc att gag atg gcc gtc act gct gtt gct aat 1200 Leu Asp Lys Ile Phe SerIle Glu Met Ala Val Thr Ala Val Ala Asn 385 390 395 gct gtg gct aaa ctatgt gca gaa aga caa tgt cag gac tct aca gct 1248 Ala Val Ala Lys Leu CysAla Glu Arg Gln Cys Gln Asp Ser Thr Ala 400 405 410 415 ctc cag cct tgggag ctc ctt agg cag ttg cgg agc atc act ttt gag 1296 Leu Gln Pro Trp GluLeu Leu Arg Gln Leu Arg Ser Ile Thr Phe Glu 420 425 430 aat gga gga gaaatg tac aaa ttt gat gcg aat ttg ggt tat gat ctc 1344 Asn Gly Gly Glu MetTyr Lys Phe Asp Ala Asn Leu Gly Tyr Asp Leu 435 440 445 ttc ctg tgg gaagga gat caa tct gac gaa cat gct gat gac ata ata 1392 Phe Leu Trp Glu GlyAsp Gln Ser Asp Glu His Ala Asp Asp Ile Ile 450 455 460 gca gaa tat gatcca acc aaa ggt gga ttc cac tac ata cac aat gat 1440 Ala Glu Tyr Asp ProThr Lys Gly Gly Phe His Tyr Ile His Asn Asp 465 470 475 ctg agt gaa attaag aaa gtg gta tct agg tgt tca aac agc tgt cag 1488 Leu Ser Glu Ile LysLys Val Val Ser Arg Cys Ser Asn Ser Cys Gln 480 485 490 495 cca ggc cagtac aag aaa aca gca gag ggt cag cac aca tgc tgt tat 1536 Pro Gly Gln TyrLys Lys Thr Ala Glu Gly Gln His Thr Cys Cys Tyr 500 505 510 gag tgc ctcacc tgt gtg gaa aac cat tat tcc aac ata aca gat gct 1584 Glu Cys Leu ThrCys Val Glu Asn His Tyr Ser Asn Ile Thr Asp Ala 515 520 525 gat gaa tgttcc cca tgt gac agt gag agc atg tgg tca ttg gcc aac 1632 Asp Glu Cys SerPro Cys Asp Ser Glu Ser Met Trp Ser Leu Ala Asn 530 535 540 agc act gaatgt cat ccc aag gtt ttt gaa tac ttt gat tgg aac agt 1680 Ser Thr Glu CysHis Pro Lys Val Phe Glu Tyr Phe Asp Trp Asn Ser 545 550 555 ggc ttc gctatt gtc ctg ctg ata ctg gct gcc ctc ggc gtc ctt ctc 1728 Gly Phe Ala IleVal Leu Leu Ile Leu Ala Ala Leu Gly Val Leu Leu 560 565 570 575 ctc ttcttc atg tcc gca cta ttc ttc tgg caa aga cac tct ccg gtg 1776 Leu Phe PheMet Ser Ala Leu Phe Phe Trp Gln Arg His Ser Pro Val 580 585 590 gtc aaggct gca ggc ggg ccg ctt tgt cat ctg atc ctt gtc tcc ctg 1824 Val Lys AlaAla Gly Gly Pro Leu Cys His Leu Ile Leu Val Ser Leu 595 600 605 ctg ggcagt ttt atc agt gtc gtt ttc ttt gta ggc gaa ccg agc gat 1872 Leu Gly SerPhe Ile Ser Val Val Phe Phe Val Gly Glu Pro Ser Asp 610 615 620 ttg acatgc agg gca agg cag gtt atc ttc ggc ttc agc ttc acg ctg 1920 Leu Thr CysArg Ala Arg Gln Val Ile Phe Gly Phe Ser Phe Thr Leu 625 630 635 tgc gtctca tgc atc ctg gtc aag tcc tta aaa atc ctg ctg gcg ttc 1968 Cys Val SerCys Ile Leu Val Lys Ser Leu Lys Ile Leu Leu Ala Phe 640 645 650 655 gagatg aac ttt gag ctg aag gag ctt ctc tgt atg ctc tat aag cca 2016 Glu MetAsn Phe Glu Leu Lys Glu Leu Leu Cys Met Leu Tyr Lys Pro 660 665 670 tatatg att gtc agc gtc ggc atg ggg gta cag atc atc att tgc act 2064 Tyr MetIle Val Ser Val Gly Met Gly Val Gln Ile Ile Ile Cys Thr 675 680 685 gtttgg ctg acc ttg tac aag ccg ttt aaa gac aaa gag gtg cag acc 2112 Val TrpLeu Thr Leu Tyr Lys Pro Phe Lys Asp Lys Glu Val Gln Thr 690 695 700 gaatcc att cta ctt gaa tgt aac gag gga ttc tat gtg atg ttt tgg 2160 Glu SerIle Leu Leu Glu Cys Asn Glu Gly Phe Tyr Val Met Phe Trp 705 710 715 ttaatg ctg gga tat ata gct ttg ttg gct ttg ttc tgc ttc acg ttt 2208 Leu MetLeu Gly Tyr Ile Ala Leu Leu Ala Leu Phe Cys Phe Thr Phe 720 725 730 735gca tat ata gcc aga aaa cta cct cag aag tac aat gaa gcc aag ttc 2256 AlaTyr Ile Ala Arg Lys Leu Pro Gln Lys Tyr Asn Glu Ala Lys Phe 740 745 750atc act ttc agc atg gtc atc tgc ctc atg gcg tgg atc atc ttc atc 2304 IleThr Phe Ser Met Val Ile Cys Leu Met Ala Trp Ile Ile Phe Ile 755 760 765ccg att cat gtc acc acc agt ggc aaa tac gtg ccg gct gtg gaa atg 2352 ProIle His Val Thr Thr Ser Gly Lys Tyr Val Pro Ala Val Glu Met 770 775 780gtt gtc att ctt att tca aac tat gga atc ctg agc tgt cac ttt ttg 2400 ValVal Ile Leu Ile Ser Asn Tyr Gly Ile Leu Ser Cys His Phe Leu 785 790 795ccc aaa tct tac att att ctt ttc aaa aag gag cac aat act aaa gac 2448 ProLys Ser Tyr Ile Ile Leu Phe Lys Lys Glu His Asn Thr Lys Asp 800 805 810815 gca ttc atg aag aat gtt tat gaa tat gca aga aag agc gca gag aat 2496Ala Phe Met Lys Asn Val Tyr Glu Tyr Ala Arg Lys Ser Ala Glu Asn 820 825830 atc aag ggc ttg acc ggg act gag ccg caa ttt aaa caa gag aat tcg 2544Ile Lys Gly Leu Thr Gly Thr Glu Pro Gln Phe Lys Gln Glu Asn Ser 835 840845 gtc tac aca ata tcc aat ctg tca ttc gtg cct gaa gag aaa cac gaa 2592Val Tyr Thr Ile Ser Asn Leu Ser Phe Val Pro Glu Glu Lys His Glu 850 855860 taa 2595 14 863 PRT Brachydanio rerio (zebrafish) 14 Met Asp Leu MetSer Phe Ile Leu Leu Trp Ala Gly Leu Met Lys Val 1 5 10 15 Ala Glu AlaSer Ile Ala Gln Phe Ser Gln Leu Gly Ala Ser Ala Pro 20 25 30 Gly Asn IleIle Ile Gly Gly Leu Phe Pro Ile His Glu Ala Val Val 35 40 45 Pro Val AsnTyr Thr Gly Asn Asn Ser Ile Ser Ala Pro Glu His Pro 50 55 60 Asp Cys IleArg Phe Tyr Thr Lys Gly Leu Asn Gln Ala Leu Ala Met 65 70 75 80 Ile AsnAla Val Glu Met Ala Asn Lys Ser Pro Met Leu Ser Ser Leu 85 90 95 Asn IleThr Leu Gly Tyr Arg Ile Tyr Asp Thr Cys Ser Asp Val Thr 100 105 110 ThrAla Leu Arg Ala Val His Asp Ile Met Arg Pro Phe Ser Asp Cys 115 120 125Glu Ser Pro Glu Asp Ser Ser Gln Pro Val Gln Pro Ile Met Ala Val 130 135140 Ile Gly Thr Thr Ser Ser Glu Ile Ser Ile Ala Val Ala Arg Asp Leu 145150 155 160 Asn Leu Gln Met Ile Pro Gln Ile Ser Tyr Ala Ser Thr Ala ThrIle 165 170 175 Leu Ser Asp Lys Ser Arg Phe Pro Ala Phe Met Arg Thr ValPro Ser 180 185 190 Asp Glu Tyr Gln Thr Cys Ala Met Ala Lys Leu Leu LysSer Asn Lys 195 200 205 Trp Ser Trp Val Gly Ile Ile Ile Thr Asp Gly AspTyr Gly Arg Ser 210 215 220 Ala Leu Glu Gly Phe Ile Gln His Thr Glu ThrGlu Gly Ile Cys Ile 225 230 235 240 Ala Phe Lys Ala Ile Leu Pro Asp SerLeu Ala Asp Gln Gln Lys Leu 245 250 255 Asn Thr Asp Ile Glu Asn Thr LeuAsn Ile Ile Glu Asn Asn Pro Lys 260 265 270 Val Arg Val Val Ile Ser PheAla Lys Ser Ser Gln Met Gln Leu Leu 275 280 285 Phe Lys Gly Leu Gln SerArg Asn Ile Ser Asn Asn Met Val Trp Val 290 295 300 Ala Ser Asp Asn TrpSer Thr Ala Lys His Ile Leu Asn Asp Gly Ser 305 310 315 320 Ile Thr AspIle Gly Lys Val Leu Gly Phe Thr Phe Lys Ser Gly Asn 325 330 335 Phe ThrSer Phe His Gln Tyr Leu Lys Asn Leu Gln Phe Glu Ser Glu 340 345 350 AspGlu Met Asn Asn Ser Phe Leu Lys Glu Phe Leu Lys Leu Asn Ala 355 360 365Gly Asn Ala Ser Asn Thr Val Leu Glu Leu Met Lys Ser Thr Asn Leu 370 375380 Asp Lys Ile Phe Ser Ile Glu Met Ala Val Thr Ala Val Ala Asn Ala 385390 395 400 Val Ala Lys Leu Cys Ala Glu Arg Gln Cys Gln Asp Ser Thr AlaLeu 405 410 415 Gln Pro Trp Glu Leu Leu Arg Gln Leu Arg Ser Ile Thr PheGlu Asn 420 425 430 Gly Gly Glu Met Tyr Lys Phe Asp Ala Asn Leu Gly TyrAsp Leu Phe 435 440 445 Leu Trp Glu Gly Asp Gln Ser Asp Glu His Ala AspAsp Ile Ile Ala 450 455 460 Glu Tyr Asp Pro Thr Lys Gly Gly Phe His TyrIle His Asn Asp Leu 465 470 475 480 Ser Glu Ile Lys Lys Val Val Ser ArgCys Ser Asn Ser Cys Gln Pro 485 490 495 Gly Gln Tyr Lys Lys Thr Ala GluGly Gln His Thr Cys Cys Tyr Glu 500 505 510 Cys Leu Thr Cys Val Glu AsnHis Tyr Ser Asn Ile Thr Asp Ala Asp 515 520 525 Glu Cys Ser Pro Cys AspSer Glu Ser Met Trp Ser Leu Ala Asn Ser 530 535 540 Thr Glu Cys His ProLys Val Phe Glu Tyr Phe Asp Trp Asn Ser Gly 545 550 555 560 Phe Ala IleVal Leu Leu Ile Leu Ala Ala Leu Gly Val Leu Leu Leu 565 570 575 Phe PheMet Ser Ala Leu Phe Phe Trp Gln Arg His Ser Pro Val Val 580 585 590 LysAla Ala Gly Gly Pro Leu Cys His Leu Ile Leu Val Ser Leu Leu 595 600 605Gly Ser Phe Ile Ser Val Val Phe Phe Val Gly Glu Pro Ser Asp Leu 610 615620 Thr Cys Arg Ala Arg Gln Val Ile Phe Gly Phe Ser Phe Thr Leu Cys 625630 635 640 Val Ser Cys Ile Leu Val Lys Ser Leu Lys Ile Leu Leu Ala PheGlu 645 650 655 Met Asn Phe Glu Leu Lys Glu Leu Leu Cys Met Leu Tyr LysPro Tyr 660 665 670 Met Ile Val Ser Val Gly Met Gly Val Gln Ile Ile IleCys Thr Val 675 680 685 Trp Leu Thr Leu Tyr Lys Pro Phe Lys Asp Lys GluVal Gln Thr Glu 690 695 700 Ser Ile Leu Leu Glu Cys Asn Glu Gly Phe TyrVal Met Phe Trp Leu 705 710 715 720 Met Leu Gly Tyr Ile Ala Leu Leu AlaLeu Phe Cys Phe Thr Phe Ala 725 730 735 Tyr Ile Ala Arg Lys Leu Pro GlnLys Tyr Asn Glu Ala Lys Phe Ile 740 745 750 Thr Phe Ser Met Val Ile CysLeu Met Ala Trp Ile Ile Phe Ile Pro 755 760 765 Ile His Val Thr Thr SerGly Lys Tyr Val Pro Ala Val Glu Met Val 770 775 780 Val Ile Leu Ile SerAsn Tyr Gly Ile Leu Ser Cys His Phe Leu Pro 785 790 795 800 Lys Ser TyrIle Ile Leu Phe Lys Lys Glu His Asn Thr Lys Asp Ala 805 810 815 Phe MetLys Asn Val Tyr Glu Tyr Ala Arg Lys Ser Ala Glu Asn Ile 820 825 830 LysGly Leu Thr Gly Thr Glu Pro Gln Phe Lys Gln Glu Asn Ser Val 835 840 845Tyr Thr Ile Ser Asn Leu Ser Phe Val Pro Glu Glu Lys His Glu 850 855 860

What is claimed is:
 1. An isolated polypeptide comprising a sequenceselected from the group consisting of: SEQ ID NOS:02-06; at least 16consecutive residues of a sequence selected from the group consisting ofSEQ ID NOS:07, 08, 10, 12 and 14; SEQ ID NO:02, res. 1-10; SEQ ID NO:02,res. 29-41; SEQ ID NO:02, res. 75-87; SEQ ID NO:02, res. 92-109; SEQ IDNO:02, res. 132-141; SEQ ID NO:02, res. 192-205; SEQ ID NO:02, res.258-269; SEQ ID NO:02, res. 295-311; SEQ ID NO:02, res. 316-330; SEQ IDNO:02, res. 373-382; SEQ ID NO:02, res. 403-422; SEQ ID NO:02, res.436-442; SEQ ID NO:02, res. 474-485; SEQ ID NO:02, res. 502-516; SEQ IDNO:02, res. 561-576; SEQ ID NO:02, res. 595-616; SEQ ID NO:02, res.640-656; SEQ ID NO:02, res. 683-697; SEQ ID NO:02, res. 717-732; SEQ IDNO:02, res. 768-777; SEQ ID NO:02, res. 798-813; SEQ ID NO:02, res.829-843; SEQ ID NO:02, res. 844-877; and SEQ ID NO:02, res. 852-875.


2. A polypeptide according to claim 1 comprising at least 16 consecutiveresidues of SEQ ID NO:02.
 3. A polypeptide according to claim 1comprising SEQ ID NO:02, res.1-566.
 4. A polypeptide according to claim1 comprising SEQ ID NO:02.
 5. A polypeptide according to claim 1,wherein the sequence is SEQ ID NO:
 14. 6. A polypeptide according toclaim 1, comprising SEQ ID NO:
 14. 7. An isolated or recombinantpolynucleotide comprising at least 36 contiguous nucleotides of a strandof a sequence selected from the group consisting of SEQ ID NO 01, 09, 11and
 13. 8. A polynucleotide according to claim 7, comprising a sequenceselected from the group consisting of: SEQ ID NO:01, nucl. 1-47; SEQ IDNO:01, nucl. 58-99; SEQ ID NO:01, nucl. 95-138; SEQ ID NO:01, nucl.181-220; SEQ ID NO:01, nucl. 261-299; SEQ ID NO:01, nucl. 274-315; SEQID NO:01, nucl. 351-389; SEQ ID NO:01, nucl. 450-593; SEQ ID NO:01,nucl. 524-546; SEQ ID NO:01, nucl. 561-608; SEQ ID NO:01, nucl. 689-727;SEQ ID NO:01, nucl. 708-737; SEQ ID NO:01, nucl. 738-801; SEQ ID NO:01,nucl. 805-854; SEQ ID NO:01, nucl. 855-907; SEQ ID NO:01, nucl. 910-953;SEQ ID NO:01, nucl. 1007-1059; SEQ ID NO:01, nucl. 1147-1163; SEQ IDNO:01, nucl. 1258-1279; SEQ ID NO:01, nucl. 1375-1389; SEQ ID NO:01,nucl. 1581-1595; SEQ ID NO:01, nucl. 1621-1639; SEQ ID NO:01, nucl.1744-1755; and SEQ ID NO:01, nucl. 1951-1969.


9. A polynucleotide according to claim 7, comprising at least 72contiguous nucleotides of a strand of SEQ ID NO:01.
 10. A polynucleotideaccording to claim 7, wherein the sequence is SEQ ID NO:13.
 11. Arecombinant polynucleotide encoding a polypeptide according to claim 1.12. A cell comprising a polynucleotide according to claim
 11. 13. Amethod of making an R5.24 polypeptide, said method comprising steps:introducing a polynucleotide according to claim 11 into a host cell orcellular extract, incubating said host cell or extract under conditionswhereby said polynucleotide is expressed as a transcript and saidtranscript is expressed as a translation product comprising saidpolypeptide, and isolating said translation product.
 14. A method ofscreening for an agent which modulates the interaction of an R5.24polypeptide to a binding target, said method comprising the steps of:incubating a mixture comprising: an isolated polypeptide according toclaim 1, a binding target of said polypeptide, and a candidate agent;under conditions whereby, but for the presence of said agent, saidpolypeptide specifically binds said binding target at a referenceaffinity; detecting the binding affinity of said polypeptide to saidbinding target to determine an agent-biased affinity, wherein adifference between the agent-biased affinity and the reference affinityindicates that said agent modulates the binding of said polypeptide tosaid binding target.
 15. A method according to claim 14, wherein thepolypeptide is incorporated in a cell membrane and the binding affinityis detected by measuring ligand-binding mediated signal transductionthrough the polypeptide, across the membrane.
 16. A method of screeningfor a G protein-coupled receptor, said method comprising the steps ofexpression cloning using an expression system which detects activationof multiple signaling pathways by coexpressing at least one G proteinselected from Gα_(olf) and Gs and at least one G protein-gated inwardlyrectifying potassium channel, together with candidate receptors andidentifying clones which yield agonist-dependent currents, wherein thereceptor is an odorant receptor and the G protein is a Gα_(olf).